Chapter 2

REVIEW OF LITERA TL'RE

1.1 Protnsn

Ptoteases. ~b Cd bydrolyw the p:peidc bonds la othcf proIrics.

con~ilutc a \ret)' Jatge Md comple'x IfGUp or ~ aad diffn iD properties suda

as 5Ubs1n'c: ~ipecirlCil)'. ac1i\'c: ,dc. ," ... ,).i~ mechanign, pH _ Ic:mpcrarun: of

acm;ry and stability profiln (Ward. 198~). They c:~ecUlC • large vanet) of

functions. extending &om the celfuLAr JeVC'1 to the organ and orranism k.....el. and 10

produce cascade syslems in an organism lo maintain horneostas)s.

ExtraccDular microbial proleases conlribuCe to the nutritional well bC:lni or

the organism by hydrolyzing large peptide 5ubstrate, into imlfllt:r m(llcculo~ thtu

'he een can absorb. ~anunaJian pancreatic proteases and inte~tin.l and ~1omuch

peptida5es ~neraUy perform a similar Dutritional roh: in the digestion and

absorption proocss of these r-pecies. Proteolytic emyme.~ are also LQ\'olved In the

regulaion of biological melabolic pruces.s.e!i sucb as spore fonuatioQ (Kllmbe'J, C1

.1.. 1968; Nonh. 1982J. spore gmninalion (Jacbon and Cotter. 1984), prote",

rnIIUratian in ~iraJ as.·iClUbly. ICUvation of ccnain vUu5oC'I of imponaace ror

~hop:mcity (KalOb et .I.~ 198~). \'.nom. ~p oflhr munmahu ren.tatton

procns (Uonda et at. 2002). blood COiI~ ftbrinotysi ... ""npb ....

Idjvatioa (Cbambcn. 3nd Lament. 2001; SUn and TsifuogIou. 2(04). pblgotyIosU

and blood pressan C'OIltJoI ell et al. 20(4).

In all cell ~)"Sk'.rnJ. ~ 5 a baJanu bet.'CCD metabolic proceun

ia\'OlVUlg protein synthesis aod bfeUdowo. and inUacd1ubr proteinasa play a

\-ita) role in the.~ protein tu.rnm'1ef proccss.es. wbK:b is CSSCDtial few tht' lkbpIacion

uf mjt..'TObi.J IInd ofhCf «Us to the new en\'ironmmtal ~ parI,,""Ularty in

7

CU\1ronmenl& lacLag in IminO acids. as It gCOCnltC'$ amino .cId pool (or ~Itbe,,"

DI newt)· ~ed enzymn md other protC1flS tHcnhko tt al • 19&4 t· "rolC'~ art:

.1t() ImpOIrtan' lor celJ d,fTcn:nIJatIon. ~lation. modulation of gene ClP'"iion.

and In ~rt1.')111&: IDI)dJfi(~tjnn and sccrrtwn (Roberts cc at • 1977 ~ Proteasn coa\o-m

InKh\"(' ~tIIn ADd 4:4ba bloS~lJ) maaivc protC'Ul moJuule~ mto then Kb"~

rorna .... dunns o.tl'llCC'Uuw auymr SCC'mion. the hydropbobtc f'C'p1t.te

ntC'ftSloa wb"h (.,lilalr' the ~ (tf the emynx through W cdJ mcrnbn.nc it

t"k>.t\Td by pro4('(1)~nc JC1UlO (Smeekms.. 1993,

.-\aa1bn, 10 the Sommc~ (" &.JmmitItc of the "t'IMIloDal LnilOll o(

Bicdmlisby ...., Molccula. geology. J'M~ 8fC' dassirK'Cl .... ~ 4 01

t;rl1Up 3 .b~~~) cll'BMH, J~) Howt'\oTr. rmub:S 00 DOe comply c-awty

*db Ibc pcra! i)'SWID of ~nzymc DOmIfDC.1.aM~ due '0 tlarar hUIC dJ"""T) of

lC'eoallKl5lr'Ul.1ure. Cum:nlly. prutca:w:::. are ~(1Cd OD the .... , o(~ .... p

omtc1l1l, • ~. C}JIC ",f K«tMlD ".wyzed. lu • du:micaJ rwurc of the ~ ~1K 'd, . .JDd

'1111 ~'Ulub(JNf) rd~ho..,Jllp * nb rdarucC' 10 !J1ULturc t Barrnl. ~ J )

Dc=pcndJnJ 00 thctr ,* of adtan. protc:aJo ~ broadly damficd mto r-o

RaJOf ~" It. cx.ru~ md ~ £.\opq"Ctdun cl~nc 1he

('CI'l. bond pro,umll IC\ the arnmo or arboxy lcnDInl of the ~. _ht-,~

C'tIdopepndub clC'3vr Jkl'lidr borKk dtsamr from the lm'NnJ of the !lub.nIe

A.~ MS the' fun~joral J'l"4' ~, at the 1("",1: s~. pt<"~ASe5 ~ funher

dawdied into fuur promlnt'1lt ~ U·,. SenD ... J'fUCCII9&!'f. laJil'lftic pn'I".~

C1-'Ucine rr.~~. and mruJloproka-.n (Harder. 1%0) Ou •• the~ ~ • rC'. rnl~(Hmcotl\ rrut~. "hJ~h di'D,-"" pnriscly fil !bID W Jtandard c~\Olfiul1oa

o..cd Oft ~ pH of then ortlmal adt~lty. prole.uo arc abo rdrm:d 10 .:-. IU·Id.e.

lKulnll Of iilLIl .. ~ prolt.uc, la the Et" h.s.'. pepfwJibC~ uc di""ldcd Ilmong I ~ ~b­

d&s~s 'omptI!omg e.,\t'PCpudases 13411·19) ilnd eodopcp'u~~ ~.' 4 ~1·~4

l.lhc:r wllh tol99.1b IIV~U 10 Tablt 2 I.

K

RrIJiruI of U,,,.,.,,.

Aceonting to the widely URd md 1DOSI cOmpftha15h'C daIabase of

profcaSn (MEROPS; hnp:l/www.merops5allsa.x.uk). aaymes or acla catalytic

type ~ ctamfted in,o n'Olu1if.llllrily di5Unct "dms~· and c:xh cb1ll b subdivided

inlo .. families" (Rawhnp d al.. 20(4). Each family of peplwWcs has hem

HSignrd a code letter dmolinllhc type of abl)z. Le-, S. C. T. At M or U for

Sftine.. C)'$1~ dftoaiae. ~ mctaIJo Of lIIIknown type. rnpec:tivcl)'

(Ra_iings aad ~ 1993). ThrconiM Iype pepCidases IN: the mou ra:catJy

ctiKO\--aat cauIytic type (~ et -I.. 1995).

T •• k 2.1 ne EC .,.rem or rlaJllftratioa of ~WMn

S"'sllbdass T~ of peptidase

Exopepti ..... 3.4.1 t Amino J)ClMidases 3.4.13 Dipepti~

3.4.14 OiJMlPcidyl-peptidaM:s I - ... ~-

3.4.1 S Pcptidyl-di~pljda!lI:!I

3.4.)6 Serine-IYJX aiboxypcptidases

3.4.11 MctaUooubox)'PC'p'idascs

3.4.IR ~ine-type carboxypcplidn~s

1.4.19 Onqa peptidases - -

udopepddases 1.4.21 Serine c ... Ijd.a.sef. 3 .... 22 CyAcine endopeptidases

3.4.23 A.sputic~

34.24 --Melal~

I 34.99 F.ftClnpqllMIaJes. of IIIJbmwn type

UI ESGpqIIidun

The aopep6dacs .. 1 0DIy ~ ,be ends of polyrcpll« c:hams. 8ascd OIl

thar s;te of action aI the Sore Icnnmus. they an: clanificd as. IkDioo .nd

,arhoxypeptidases.. n:spcdi¥dy.

2.1.1.1 .ualaoprprfdata

Am.inorcrctdti.n Kt .ar I flu S'(mn1nID or tk pol~~ dWa aN

liJerak • ~ anUno Ked Iftidut •• ctipcp:* or alripqMktc They 4ft ~-. 10

mDro)\.t' ttxo ~·'nmin.aJ . Met' tb.t may be found Ln ~~~}) C' .. ~d

pmk'UH t.ur nl'C m milll)' n.mnlJv ~-.:urn.., m:IICUtt pIIOCt"lIn. ~

ottur m:l .... \arJf'I)' ur nn~t-I.I ~C's nrlu.:bn. NttrtUI and fun,; f\\,tal.Vln.

14:6) In grtaal. ammnpqM~.-r inlracdl .... ~s.. hullhm:- ha been.

smIle rrpon on .n rll:tDcC'lIuJar .minOJXPljdn~ pwduC'td by ."~III&J ory:-iW

IUbbc et al. J9~·H. T1r 'IoUMnIC' IfI'K"Jk-.ltCS 0; die c:~ rrom ba.&:ria.nd

run,. Me dastlfM.'lly diffC1CtJl In thld thr arranisms can he ctiffcrt'nu8.ed or] thr basi!

oftM poroduc l profilcs oft'11Z)lJX' h~droJr't~(Cm1)', 19181 Am&n~Mia.c llTom

&"herid.'1I {vi. i~ • 'lUll: prou:.~ (4,00.000 Dll It h~ I broad pH tlpttmum uf

";'.~ 10 1 0 ~ olnd fCt.tUlfCj M,:' or \in:- for Optimal J('u"'i1y !M4f1.;~9 ADd Old; .. 197A I

ne Hlle.//Ju /idJ,·"t(or"tI.V Ilmtnt.i pc-ptidtic hll§ ill mot«ulAl' wctsht of 34,000 O. 1.

COIIt8.1nJ I R·.t(1m of In,'' f'Cr mol. Ind it!. .Cllvtty ,~ cuhaacc.d by Co:' IU"'" On 'he"

othtr hand. amanopeptldu..r rJ from BQrlIJuJ sltlUo,h .. ,.".('fth,/tn as a dinltT w;lh I

ml~rcullU wtight of ~O.OOO co 100,000 Jh and is a1Ch\'.led b}' ln~·. \4":. OT C(J··

tCO (Sloll cuI. Iq'6)

lll.1c"~~

1k nr"' .. )'pq'C~ ac-I it C"'C'I'1TUI' ... J~ of 1k pol~ chain .d

11bcntc ~ mtgk ammo k'ld Of. d.1ptpclde C.rm)'pqJltdbe. can be d.,,'wkd IDIO

~ tMlot p~ IWPxly ~ CIItJo:IL)"J'C'PKWn.. mdaUP(ubot~dasts.

aDd C)~nM c.ubcl"~'pep4dao;,eS. b.u.cd 00 sbe IU.turc of tM anuno aci4 rrsao:iuc:s at

Ibc acllVC W u' dIf: mxymo. Thr scnne ~)'pC'ptJdMes it>alllkd rn.. Pt'ffu:u;n.", spp. SJcd''''~'''nn''.J "PP, met .fJP"K'Jiau 4'P .n SLrDllG ID Ihc.~r

stablUr\. molecular we,~hl.nd crfl:'t!tufinbJb1lOG, MtblJoc.arh,."'l>-prptd~, frot:1

SaC'dtam".~'('t1 tpp, (Fell, .ad an .. dlel. 14166) and rv-h"".HIIU )PI'. requu,

10

z.n:. or COl' for their activilY (La Cl at~ 1969). ~ C'II:Z)'IID QIII altO hydrol)'7,c lhc

pq1Iides in ... hich dJc pcpbdyllfoup is replaced by a ptcroyl moiety or by acyl

poups.

EodnpepidaMs an: data:lerUcd by tbcir psc:fcttntial action •• the pepcNlr

bcxKh iD lbc Umc:r rqionI 0( Ihc polypeptide chain a\\ollY &om Ihe S ~ we· tennini. ne preseatt of ~ fI'u ammo or carboxyl group has a nqative i:I:I.I1ucan:

OIl c:ozyme ac6vil)'. ~ eodopcplidue~ 1ft divided mto four subgroups baled on

their aaalytK mcc:hIInim1; (i) 'LTinc prolcata, (ii) aspartic pruICMCS.. (iut C)'SlCUIe

proI~ and (i,,) mctalJoproleul~,

1.2.2.1 Serine protease,

Serine proteases are chanl,,'terla:d by lbc prQC.:ncc: of a §crinc: group in their

active site. They are numerous and widespread among viruses. baclen" and

cukaryotes, suggesting that they are vitBJ lO the organisms. Serine prolcascs arc

found in the cxopcp1idue and endopt:ptidase groups. Based on their Jtructul1ll

~imilarities.. smn~ prole.ueS lulve been grouped ioto 40 families, which blve I1ftn

further subdivi&d into 10 clans 'With CODlDlDfl anc;;eslun (elm .. -... 1994) lk

primlJ) mucnns of the memben of four cltos. chymotrypsin (SA). Albtilisin

(58). carbo,ypqJCHIIic C (SC). MId &:MridUQ D-Ala-J>..AIa pcpbdaK A (SE)

~ CoC&lty WftlaIed. sugntmg 1JuI tbm: arc at IcaSI fOtB' seplrah: cvnlutMWrolfY

ongms. for ~ prolc:a5C$. Clam SA. S8 and se ba\"C a C4JIIUIIOO IQC'tioa

mechanism ~ oC. ccaunoa caaaly1tc lriad of the 1hrtt .mno Kids.. tCriac

tuuckuplrile). ~ (clectraphi~) and lus1idioe (base). AJtboDr.b Ihc FCJmdnc

ori~ of these rmdun .re 'imiJar. Ihc proIcIn ~ ate qWIe ddremn.

forming • Iypial eumplc of. convergent c:volutiOl1 Tbr caIalytic mccbmisms of

clans SE 3Dd SF arc dlstinctly da tTaaJt &om tho!..e of clans SA, S8 and S~ since

lhry lack the das$ical Scr·Hi~·AIJP trt.d. A.nother tn1eftirins fnlUre of the same

11

prouoases is fae ~ioa of ~ residues m 1bc vicinity of die aaaJybc

...m~ raiduc hJ 'Qnu IJx srairOIy-XU~-Yaa-GJy (Brauxr. 1988).

Seriac ~ Me rccogniad by Ihci7 irrn'asible inhibilion by 3.4-

dichbui1lXOlmlriD (l ..... OCI). L-l-arboxy InB·U-c:poxYJlR'P)'I.~

(~xtiDr). bu1abe (E.")~ di-isopn:Ipylt'ha~c IDfPl. pkoylmcchybWfCMlyl ftumdc tPMSf). IDd lOsyl-L-fyWlIC cldoromnhyl tdGor mCK) f~. 20(1). Some of the seriuc procc:an ~ inhibiaed b! Ibiol

reag~nlS sudJ as p·chlor~uribcmoele (PCMB) due to Ihe prcsaICt of a

C)S1CtDe residue new the k1M sete. Serine protca5e5 are ~Dy KUve •• xattal

ani a1kaliDe pfl. with In optirrMm1 between pH 7.0 DDd 11.0. They ha\'e broad

smsualr ipCCilicitif$ .nclllding esrcrolytic and amsdaK activi1y. Their motecut.r

IN.';..II range between t 8 DIJd 3~k.L>a. and the serine protease rrom Blaus/ea mspora

has a molec;ulaf mass of J 26kDa (GOYind et al.. 1981). The: isoeleclric poinL.4i of

serine proteR~eN ar~ gtmClrall), br.twecn pI 4,0 aDd 6.0. Serine 111kalint proteascs that

are act,vc: id hi~ly aJkaline pit rqm:sent 1be largest subgroup ofserinr pr~.

1.2.2.1.1 SeriM alkati_ proaeun

Serine Itlkaline pmlCaseS ~ produced by toCycnaJ bactai". mo1ds. yca.«. ..

and fungi. Ttqo 1ft 'nbibited by OFP or • poI»o pmCcasc iDIIibd« but Mf hy

l05yl-L-phcoylalaninc chlonJJnelhyl kctmt CTPCK) or tusyl·l-Jy~inc thlorotJldhy'l

k.done (flCK) TheIr ~ specificlfy is similar 10. bullcss m.ftI'CId tbaa Iba

of cbymobypsia. They ltydroJyzr ~ ~idr hewl. which has fyn:Ia1Ie.

pbcn)JaIaDJDco 01 ~inc .. fK' carboxyl silk of the spbuin& bood. The optImIJ pH

of albliM JJfOkaIe' ~ cuumd pH 10.0. amd 1bcir isodearic point .. .round pH 9.0.

Tbcir molcxulu tUB i, in Ibe ~ cl IS '0 lOkfla- AIthou@h aJbIiac seriae

pFCJIraJfto are pnduccd by ~I badcr.a §uch as A,tAt'Ob«tu. SlrrpltJIIIJ"rtl _

F1~niuIII 'PP. (~ et aI~ 1983). subtilisim ~ by &Jcil/Ju

spp .• ~ the bo1 known AtIcaltnr prolcaSeS an- ako pruduc:ecl by S. ~JUK

12

(\t.izw)o Dd M •• ~. 198" ~ IDd rt.laaJc:mow. f'uop web om C~1d Ipp

~ et aL 199)., . ..f.spr1Jp/hu ..... Nn.roIponupp. c LU.f8 et al.. 19ti1 t.

1.2.2.1.2 Subdllsiu

Sublth'tt~ of lJoclllus orig.in rcprc .... the lCt'ond lmgeu rJU2.d~ of JCnne

protcases. rwc d,fferrat Iypn of alkalmc pru1C.Jt~. f,ubuUs.in CarbbcTrg and

.ubuh~m So\'o or blC1crill proIQSC ~.gaJ1I...e ttut N9) hlllw bc~n ulc-ntifilld

Subuhsin OU'I*rg PJ~u('ed by B~-,[J1J.f IiCMnI/orntl!1 wu disoovrl'!'d in 1941 by

L1adcrsUom. [.an, and Ont'SC'D " the Carbhcllt l.hotllOJ)- SubUb~ln No\'O or

BP~9 $ Jmld\lccd bv &Jell/.d offf."loJiIlwfari,·IU. Subblu,UJ Carli.bag " WNicly

used In dcotcrgcmr.. Ib umu.1 produt1ion amounu to aut ~OO tom; ofpllrt' n.7.)'me

proecm. SubtiltwD R'~~ •• Ins. (QiiJXn: •• IJy ImpgnaDl Rotb JUhbI .. m, bw •

aDccuIar maa of 27 ~U>a ha differ ina =-cb oL'IU b) ~s CDmD." Tbty

~ UD1* popcn!n ~ ., aD upti:aa11ca~hft' nf ~c: and Ma optiaaJ pH

of 10 It 80Ch C'DI)'UICI cx.htbil bn.wi substnle 'PU1frci1y .... M\-'C.,. actn.'C'~'1e

mad made up of Scr:~ L th..64 and A"i',U. The C-.vl~bn-, CAI)'ftIC haa hoadcT

,utHmtr !ip«".tktl)' .nd ~ ftllI dt:pmd de C.:· fur us st.bili~ """ ac:tNc 'ri~

confotmolflaa o( subt,la~ r" ..anrw to ... o. ~ and cby~n dapJk

lhe ~ilanty in then cn'eraU moln:ul. lItI.n~n~ The ~nn~ .U.allDc

p1"()4ca.Qe from lh~ fun~us C(I,uaiobulus CQrmlolll.f \\0" sbow,. to pos~C'\,. LI ciuticw.ll),

diff",cnt :llru,'"~ frtHn 'luhrfhsin Carlsbcflt in !lpit~ uf their funcri<lnaJ 'oIlmtlaritier.

cPhDdlwc etal. 1997),

A5p8nX actd proteases. communl,- UIO"''11 D. ..di,. rrotn~... .tr lhr

~ tbaI «pcad ca .. ~ aod r~' !ut then nlah.uc .-twit)

Ac:tdtc ~ ha\or bfte S'fOU.PI'd mi(l ~ rarnfl~ natrId!. rep'" (A 0.

~ (Al) .. ~ftZ)'IIr' rrom ... ctrOVlfU50 tAl) aDd bau bn-n plkCd 1.

clan AA ~ 8irt'1t. J OQ~. ne JTlC'tcben oi r.ftub~ A l ad A: .t lmo\lol1 10 be'

ttlattd to e«h OIbn, .'!uk thow of famtJ)" AJ ~ tome rellledDeu 10 AI aDd

A1 MOJiI uput" proec:a.r.~. "'"DrIll K1rVII) .)0..- pH (J 0 to ".0) IDd ~

itoC"«ul(" .,."ntsln eM,.. of pH loO 104,~. The" moJccullr mass is iD die IaDfC

of 30 '0 45U>. lhe mt'mbns of the pcpsm ramaly have. btJobaJ SU\l(IWe WIth the

ICtWt-l1lc deft located het .. "" tM lobes tS1dcd.l a al. )qQl) 1br actn~-'SI1C

ISput~ aud ,~duc 11 ",ralollt'd .,thm the IDOflr ~Xu-G"'. m ... lhcb Xaa can be

'5a" or 'Th, ~ .~n' .. JIIulrun IR inhibi.ed by pqKtat.'D fFilqnaJd d 11.

1990) Tlk-y Itt .t$O 5CnS1'iw 10 diazoketooe compouads sudI lIS dloUOiICdyt·DL·

DOfI~nr mt'lh)'1 tut', fDA". aad 1.2.qJM)-J... fp-nurophmoxy) ~

~EPf'rI,". III die ~ of ..:~r JOD$.. ,.1rcrob1al Kad prOlQSn e-,habu spedficll)

'piM Ill"""'''' or bulky .mUll) acid Tl$idu.c1t. M both s,de~ of the pqHlde' bond,

whlC'b ,,. Mm"", fO pcpJtn. hUI lheir action it. less ~tri1lgent 11,*11. that of J'C'P,in.

Microb~1 n'p.n1iC' pro4ea\cl nan ~ broadly di"'ld<:d into two MfouP'" (i) ~in·hkc

m7}'1tlt'S produced by . .f,,~'gl/JIIJ. Pe"fc-ll1l"m, RIII~oplIS and N~ro,tpo'iJ and (u)

re'DJun-hkc enrymes proou&:cd b~' £ruJoJhia and Mucor spp.

2.2.2.3 Cysl,lnellIIlol proft-asH

('''''Sl<lM ~ oc~ur ID both prokaryolC"S and tUUt)'(\te-" About 20

faIruoo of cy~tt1llC prOl.elnon have been rec~ued. Tbr -.::11\ 11)' of .n cy51l"lftC'

proIca..~ dqcods on • Qt.IIytK dyad masiJlmg of cysrCUIe and tu"'dinc. The

ORb aloe),,' IDII 'Hi!' CC'y)·llisi or His·C'ysl rnJduct, differ. amtKIl tbr flmilin

~o.rnt. 1"""1 ~n). qlotCIlJC ~ MC ilCU~ only m ~ prcKDCC of

rechaca;, 41D!t~ ~uch ti H'" nf ~y~ ~ OQ thnr ,$de .:ha," ~dic\1~. thc) . .IK ",.wtl~ di\'1dc-d mkt r,'iIt pvups fl. Pll'4ln-lW:. to) IF)'l"oIn· hK ,,·lsh

~ far dcna!r .. the- .....-~. (Iu) specifIC to .Jut.nK .ad lad

(1\") othcn. PapaJn t.S thr ~,~n C}~ prolCaK- Cyslrlllr prouun bavr

~ pH ~ .altboup .a le.- of them. t J. J)~'" ~ an:

tnbimaJly .I.:twc al KldiC rU n.cy Itt ~~bk 10 1oU1~"'CIr)-l .,c:nI$ web .,

Pl.""MB. but In" unaffCl~kd t-v DFP and rht'lal·dlrbdina..,nt (1mtnpa.n.

14

produced by the anaerobic bKmium Clostridia"" l"stolyIfOllff. e~hlbtu a tdrmpl

specfficJty for argiayJ nsiducs at the tarboxyl side o(the splittina bond aud differs

&om papaio in ilS obligate requitemem fur calcium. SuqMopaia. lbc C)'IIftDc

prDtea5e pmdnCM by ~ spp .• sbow3. a broIda $pCCiflCily. md ....

oaidized insulin B chain and odln S)'IIlhetic 5Ubsbw1e$. Clc»aripain hM an

iIoeJecttic poimI 01 pH 4.9 and • molecular mass of~ .. iIerai IM i'JOCk-ctric

point Md moI~ mass ofstreplopain eR p18.41Dd 32kDa. rcspcctivt"Jy

1.2.1.4 MdaUopnlnses

Mefallopmtea5eS are the mast divnse of the cal&lytic rype or pro4cue-s

(Bardl. 1995). They Ire ChDJ1I~'Cril.ed by the requtremenl for 8 dinlent metal um

ror their activity. Tbc.')' include I:nl)'mcs from a variety of Of IS ins such as

collagenases from higher organisms, hcmorrhagic toxins from "lake venol1",! and

thennolysin frem bacteria (Hibbs et ttl .. 198.5~ Okada ct al .• 1986: Shannon et 11 .•

1989; Weaver et ilL. 1977). AT present, 11letalloproteases have been cJussi1i~d into

54 families (brtp:/lw\\"",'.mcrOPY.!lHDgc..'1',l.Ic.uk). which have beet! gr\'1uJ)Cd Il1to 16

clans based on the nature of the amino add thal completes tht" mehll-bindjnlli1e~

c.g .• clan MA has the sequence HEXXH·E and clan MB COIT~nds to 111~ motif

HEXXH-H. In ODe ofebe groups, the nxtal atom binds at A motif oLhcr chm the"

usual InOIif

Based OD 1be !tpQCjfJcify of tbcir action. meulloprotcases can be divided

iDID four groups: (i) neuual, (ii) alkaline. (ui) U)-xobacsn-I and (I,., Muohut'l~ U.

1K neutral proteaM$ :ahcrw 1ipC'artcily for hydrophobic amino ac~ whale the

alk.a1ine prorea5n possess it Yl1Y broad spcn6cJty, JI.\'xoNt'tr'r ~ I 1.1

specifIC for small amino acid rcsidues on either s.idt oftbe de." .. gr bond, .'bans

.~ pmttase 11 l~ ~lfic for lyw.oe rc:s:adue oa the Il1W1O SJdc of ~

peptide bead. _0\11 of them .re iabibi1\.-d by cbdalin, agents fo.UCh as fOT A hut not

by suUhychyJ agentS or DFP.

IS

ThmnoI),&Il. I ncvtnl proens.e. is the IDOS1 &boroughl) c:hanIct.erized

mr~ of clan MA. HI5udlllC rn~.es from the HEXXH motif K"n'r ill z.. lipnds. Md G1u bat • nataIyt.c fuactioa (Weavt'f er al.. 1977 •. l'bamotysia

produced by B ItrtJnH.~"1II is a single peptide witbGuc disWfwle bridges'"

has • mo\ccut.r mIU of ) .. UJa I1 COGIatJU _ n.5elluaI Zlt 8lm c:rnbcdded ID •

c~ forrnrd btrwrc r- 0 f<-Ided lobes of _ protml lad four Ca aIoDH. ..met.

impan wrmll6l.bllil) 10 the J'"*Ul. 1"bmnot)oslD.' a wry stable ~ wiIb.

half·lifC' or I h.1 aooc

Col~C'. ~hcr IrnJ'OfUDl metafJoprou:a~. "'M ftnl dn.:O"cmf ID lbC'

('ulturr broth pf tht' &nIfll"bK b.ctmUla OOftrtthlllll AJJrol.\tln,. ... .,. • CClmpwc::uf

ofto:uc prodUCb uter. It You found 10 ~ produced by tht- ~1r bacmimn

AcluolffOlkllU" IOpAdP .. nd other mic:roorpm~ mcludin, run~i (Drmina and

Lyscnko. 19%. The' Irtiun nf l:'ollapoasc ll\. "er), '4pccific; tt, It ~'lS only on

rollag(n and gelatin and rK'1 Hn any of tht other u'JLUll protein "\Jkl\tratc~. Efa.slA.~

prodtKcd by Pj,t'utkm...",a.r at'TNIt"WSfI is anotbtr Importan' mcmbt'r or tht' neutral

IlEtaJloprolc:.bC ranUly.

Tb&.- .1Ul..UM: mctaUOfIhllCbe'i. produ.:a1 b)· Ps~uJomflll"., ~.·'~!."1I0S0 iIIId

Sin-ana "ftP lie .. '"1h~ In the pH raDJC' from -: 0 Iv I) 0 and h.\c mulcnwu m&j.S In

Ox I\"p<'I1 ot 45 to 6W>a. M~:wbQ(t~,. protC'.1iiC' I bas I pit OpU111UU1 v( 9 0 and 3

mokcul., rnau uf 14kDa and can lyse ~U w.n~ o~· AnlrrohflC"ln ('n'¥~lIopo1la.

",-brtnl pmtCllW 0 c:aoot ly'iC che bactcriaI f;CU,,- \1auiJl n1CUlloprulC"aKs pla)" ~

pronunC121 rolt' Ul the dr~k.~ of the ntn.:dlulM matn, dune. I1nuc

~~UI. dtffC1'e!1UMk'Jlft .lnd Vo·OW1d bc=.tltll &net ma)' k u.cful ID Ihc

treaDilCf" ur d.~ SoUth a\ cancer aad anhritlS .8rCWtltCl"C'I al. IWS)

fbc mcch-num of aiM-m ~i pnM~~ ~ hem A wbJtcl (\f ~"fcas m~n:J1

to fnCin('ilI:n and 'S.n'CRl fl"J'Ofb ate ."tDlabk m "I~~ ". tc'rmc protease\.,

tipU1JC pmlc,"",. mc1.IIUo"nl4l'lK('!; and cysrcisx- pn.'f~_

J6

U.I Sni.e 'roceawa

Serine proIcascs usually follow a nwo-stcp rradioo ror hydm1)"'!!'. in which

I CO\"3'enlly linked tmyJOr..peptide iaknncdiatc is formed "'ith tilt Jow. of IM

UIUDO acid or pt"pIidc fnagmml (fastre7. and Fershl.. 1973). Thi~ acylauon Mep ~

followed by a deacylI'boa I'ftl"" ... htcb oa:un b} a nudoophihc .ilIaC" on ,~

incmncdiate by "''UU. ftSUhq m bydrol)3is of the peptide (fig, 2. t, Serine

radopeptIdascs can Ix cbssif1Cd. anlo thtce fUOUPS mainly based on lbc1r pn11W)

",""ratc pttf~: (a) tr}'P'Jin-llke.. whu:h dea\-"C after positively ~

rnadues; (ii) chymotrypsin- like. which clca\/'eos after large bydrophobic n:sidun;

..00 (iii) e1a...tiI5e-likt. wh.ich deaves .ner ,man ~c fc:s.idun. 1K PI

raKlue c.clu~i"'cly dictates the site of peptide bood cla,,-agc.

"-...... ........ ... -c-C-·-c-c ...

.. I 11 It I I '~f . Cl .: 0 ~ ..........

... .. - I --

1 1\ I o-.. --.~.,.,...-.. -.-,

o

• ~-e-c-o· It I •

., 0 •

... 1 o

l • ... -c-c .. I ,

..... I .. - •• .)1--. -o-c

~ . o ., ..

.-c-c ........ • I 11

• 0 f ~ ..ArfllrtI ..... c ... • ..........

• "1 •• 2.1 Schcmabc ~ion of the stcp6 involYtd in calDly,is

hy the :scf"ine proltillt type of enzyme ...

17

The pnmuy spcc1fl~11) Ut .ff'ccIi:d cnly b)' the Pll'C14~ the K'&1dues •

0lbc1 powtioas .tJm IM "* of cia, •. Tk sub Mlr IIII:fKlklQJ 3ft' ~alizcd 10

lIfJttlfK: anuoo «Ids .round '" PI ~ 10 • UDIIfUt' sec (J( SClqueKCS CIa lhl:

Some ar lite MnhC' repIidnn from ,.fc~~ 'PP. GC' lY'lIM-JP«1rtC

~ fMa:s.aL d.t. J~~t ,,·bmti ~ from Clanridl".,. ~ ft' IIJI.DIDI=

speafic Cclostn.,. .. , (Glib rill.. 1919) ... those from FJamboct~ ~ ~

post "uhM'-spt'tiflC (Yl~" ('IaL 1980t Eadopcptidurs tha1 atE' '\pcafk 10

8lutaJnlc K14 cd aspan1C Kid reiltiua hA\'C Abo ba:a found In 11. Iri,.",ftH1ftlS

IIIId !llIplnllDC"tX.-., ftl'l'tl.J (Dnl'C8u Cl al .• 1972)

lbc r'C'ccnl studlcJI based on the thrcc~imen"lIlnAllib'\.lctura I)f prolea.-.es

and cl.lmrari!lollS of amanu acid scquenc~ ncar the pr1m1J') \ub~'nt~ ·btndina lIitt"

In tT)i'5tn-likc prl1l~!e, uf nral and bacterial OOSIn rolllBut a puC.UVI: genC'raI

substratr bincUug "ClMmt' for prnlca~ with specifid,y towaro. tJutamit aud

Im'DI\U18 ... hiihdmc fCSI..1L1(, and I b)'mcu.yt function. Uo .... 't\·tt •• rt'W olher SCfU1r:

proteaSb ruc:b as pt'pti~ A from I::. toli .nd 'he ~prC,ti.iWIf 1.('" A iIlKIv. t.iiiotincdy

dltTmnr m«hllus.,m of Ktfun ,,1thou1 the c:la»te Scr-H~AJp tnad (Bart'lt, 199-1.

Sm:n&: ur lhe J'f" me ~ .rf ~ in lhc ytoait)' of tbt ceta.lytje sc:rinC'

ttSldur. t1u1 Ibrtr cud POU1:JOM arr ,,'ariablc I Brenna. 19111). The th)"I'aOtI"ypsm.

Itkr t'nzymr5 are cunfiDcd almost t'Dlm:1y 10 aimak.. \hit QCeptMlM bcl~ lryplUt­

hle- CDt)me~ 6on1 «tiauru~~C1~s antS Slxrh4rgpo.)~ 'i'P and from Ibr (UD~"U"

"'ldQramt u.J~ptJnI •.

:\ fa.' of tk scnnr rro'Q .. t~ bc1ocf.nJI10 tM sabnh~ln fUul) ~ . .&

aulytl< .0Id ~ uC ... MmC rHJdun a. in the cb~ raa.Jy;

bcr."C'\:J. tM 1't1.due, OC~Uf In a d.1JcTcn. «dt'J' r..up.HI)·Snt S«nr nrmbcn of

lbc: sul1lbuu fa:nal)' &om ,he TrlflTlJcft.WJt .... M~/II:I"IrI:I'" spp fftlUt~ lhiol fOf

tbctr .,t."lI)_ T1lr llUol ~c 15- annbutar-k It) Cyst'! nc.v the .at .. th't-~11J:'

hl'SEjd'I'.t' C J.m} (:t .. d . 1986 t

18

The carboxypeptidaies are uousuaI IIDOng Ibr serinc«.-pc:odc:o1 ~

'ia m. lhry are maxllDlllJy Kti''e at acidic pH.. Tbac CUZ)'IDCI are bowII 10

pouas a "GJu· ~ pRUdina the catalytic '5a'0. which IJ bdanai 10 be

mpoIlSible r« their 8C1dic pH cpImIUm. AJ,bough tbr ... jority of the ~

proCeaIn (W'Caju the: caW)iic triad Ser·Hi. ... Asp •• few use the Su--" ant,.ac

dyad. The (jhHpccitic ~ display. pronowx:cd prefc:n:uce (or Gl.,.X.

bcabova- Asp-Xaa boads(Ausiew cd Smith. 1976)

1..1.1 A.spartit Prot ....

Aspartic cndopcpticlases depend on lbe aspartic acid r~jdues (tIC their

cata1ytic acti,,;ty. A prral base: aWytic: mrchanism tau bet-a ptopo5ed (Of Ibc

hydrolysis of pmCctn."" by bpani~ prot~~s such as penkilWpcptln (lames et al .•

1977) and cndocbiapqJ!in (Pearl. 1987). CryslaUographic studies have fib"W11 Lb., the enzymes of the pepsin fnmUy are bilQbt..'d moJecul~ with the ltClj\lc lite ctcfl

10C111cd bCrh1:en the lobes and oBch lobe contributing OIle of Ibe pair nf u"pDrtic

fW.:.d rcsiducs Ihat is essentll11 for the calalytic Ktivity (Blundell cl Id.. 199 I;

Siclcdu d al.. 199]), The lobes are hOtnl)logOU5 10 one another, having .filCh by

lene duplkadon. The retrupcpsin molecule ha only one lobe. \\·hich carries only

0I'le aspartic: rc:Wdue _d the activity ~qui.n.'S the formation of • noDOO, .. 1cN

homoduner (~iUer d al .. 1989). In mos& of cM CllZ)'IDCS fmm the pqtsia (lmi1y.

the c:alalyt;c "Asp" raiducs a~ comatned in an Asp-Tbr-(1)'·Xu moIifia both ~

S· and C·feImiIW Colla of the enzyme. where Xu IS .~. or ·Ihr" .... ~ sjdr

chams CID fonn bydrogcD bond 10 0Mp'. ~. x... is. ·AI.' In mMt of1ht­

mropeps.lnL A marted c~ion of cysteine residue is also cvMJ..'Ill in ~K:

proI~ The pepsim &fId the l1taJCM1fY of GIber members of lhe fanuty ~

sprci fKily for the doaq~ of bonds in .. ~ of at ka:a1 ~l. residucs with

hydrophobic amino acUis iD bodI lhc rt .00 Pt9 positiom (Keit. 1'192). Tbc

"JI'.,;(icity of the cata1Y'iolS has b«n cxpa_ined on the bb"is of .~i1.b1c cryr.W

~J (liu et al .• J 996). The SoINtturaJ and klDetic studies also h.t,~ SUgc51cd

19

C..".l

that the mcchani5.m hwolves general acJd-.base catalysis with lytic Wlter molecule

lhIl dirmly putitipllQ in tbe fCICIion (Fig. 22). This is supponed by Ihe aysW

sauduro or various »partic pn1If'aSe-mhibilor compIaes ... by che dUoI

Wnblton mimicken, a tCU'abcdra1 iMmneddle fonncd after the anack by the lytic

waRt rMlccule (JalQC'S et 11., 1992)

10

Po

.. " .... -c· C--iI N .t

.. fill t ..... -c-~- .. -c-c ...

.. I ',I 0

_0_ . .. .. ~ I

..... .Ao __ o

- ........

'0 ,0 .~v ....

o .. I

III

C ,-c'

", o )--= ..

It c

P,

.....

o

.. , Q 0 ...---

. ·"'0'·" I

a 0

/'-.

0 .,' I

, , ~. ..... ~i- ~ ...

\

0

o ' \. •• ..0 .-

0

..

VIa. 2.1

(A) Schematic representahon of the genera] acid-base catalytic mechunh.m

of the aspartic protCltse: type of enryme.

(8) Schctratic rcpreseat.ion ur Ibe multib1de of hydrogen bondinl

mlCfadions available in the active ltile cleft of a typical Hf'I'1ic

protease:.

21

--i- ,-{ • -Ill -_""'*

CIJIt. I .... I ,-( .. ---.~

-. -.. · r-< .. - .. ~.~-...... • • .... -c- c-.-c -c-e

.. I 11 • I r _ 0 p- • r. •

-. ~ rl

-. --I r-< • "--",- ...-J ..

-c-c I • '. • ne ,'-pw • .'

• '. w_t�_- ~ I ! ""'V'" . · .. .... -C -c-'-C -I"

~ 1 .. r Ji. ... as ,. '" I I • • I \ " •• 1' ........ . ..

I t -.- ",--... -. fil. 1.] SchnmtK' T~SCn""Klft oflbc *'PS In\'Oh'ed In t.~""1\

by the cym:~ne JlrQlc'ast' type' of CIIl')111C-

CY'ltUIC pr01U~ ut.ll~ the h)'dro~~ of cartlo.ybr; K,d dnWolb\"e'J

'hrougb~ ~ otispla,(mnu pMh.a~ m\vl"wJ,maaJ aCJdt-.~ fOlTTLlllun and

h~'drol)'S" ul _ .cyl-,h"" ,nIenntdi*_ TIle RlC'ft..mn of If:IMJD of (~

JXCI!~ It. dun'~ 'nndar h.l th .. (If ~nt P'~~a.~ A t.ttl1mr smnlanl)'" Use

cft~l:d III thr ~khon mrdwrum f<N' ~U~ ~~ ~r d.tTm:nt 4:'\'oJuuorw)

dO~- Tht' pJ.tt pc:pIida..- papain ('.1ft he C(JrbJdn"ed 1be an:hctype ot ~cirJc

ptpI.~ and ,C1MtitutC'1o. tilOd rnndcl (N tbu tanul) "f ~'mo Th~ u.t .. lyZl!

th~ b~drob~u of peptide, .. milk otCf. thlol &:$kf .and tbIOO" ntC'f bonds (f~()I~.

I'NO) Tbe in .... f IIqt iU .hC' ,,-atalyrlC procels (tiP; 2 H involvc\ the IlOflCO\1l1eDt

22

biDding of cbe free tmymf and ~ ~ 10 form the wmplcx. Thil is foJlow.:d

by tbc acylalion of the aIZ)'IDC • .,jlh 1hc formatioo _ release of thr first product.

tIx amine R9-NH:. la .. nest dtaeyJatioa s1cp. me acyJ..eDZ)1DC racu .11h •

WMcr molecule 10 Jdcase thr KCmd produd. with the ~ of rree eta}me.

TIlt enzyme ..,.m. coasdu of a siDtfe prvtejn chain folded ID rona rwo cIomIW

oxaininS a deft for Ibe subsIrate 10 bUd 1k ay5IAI SlnIctUR' of pIpaZ

conflI1DCd Ibc C~ His 159 pairing (Balcc:r and DrmIh. J 98~ The praoenc:e or. amervcd aspll'PM; n:liduc (Asn 175) ID the proUniIy of catalylic hiSljdiDe

(HislS9) creating I Cys.His-Am m.d in ~_ pqmdascs if, eotWden:d

anaJogou..~ to the Ser-His.Asp arraagemfQt fOWld iD serine pr~

1.3.4 MmUoprolasn

The mechanism oraetion ofmc'ltlloprotc:a.o;es (Fig. 2.4) is slighlly differenl

from that "rthe above de-scr1bcd prolcl!Ises. These enzymes depend nn the pr~!M:nce

of bound divaleor callOUS and oan be inoctivlccd by dialysis or by Ihe Iddlti(m of

c:bel.ting agenls. for lhcnnolysin" bascd on the X-ray studies of the complc" with a

hydroxamic acid inhihitor. it ha b~ propor.ed that Glu 143 MsisfS tb('

nucleophilic attack of a WIlier mol~ir OD the carbonyl carbon of tJl(, IClwle

peptide: bond. which is rotuizcd by Ihc Zn!- ion (Holm and Matthe'ws. 19fU). MOAI

or the metaJlOJIRl'asn are nuymn ODataining the His..(jlu-Xa·Xu-Hls

(}fEXXH) DJOIif. -iUCb bat been sbowo by X-ray crymlloggphy 10 form a J*'l of

&he site for hind.ias oflhc metal. usuaJJy zinc.

23

..... -1Iil'- ..... 7-\ ~

.-~-

~ )-" ~:;-.~ .... ..v • I • CIO XO Y-

I

~, ... .. ~

Fic- 1.4 Schcll1,gtic rep1bCnt&lion of lhc catalysis of ~ptjdc bond cleavage calTied QUi by. member oflbc mClalloprolca5C c1au of enzyme.

In gcncn1. studits of tbr ~baDism of action of proteasc5 have revealed

tlJat they c,.hib,t different l)'pe1I of mcchanir.m bucd on their active silC

coofigumioo. The .c:'rioe proteasel contain a Ser-His-Atp catalytic: lriad. and the

bydroJ)")ilS of the pt'pIlde baad aD\'Oh.-a; aD acylaUOll step followed by • dcacyJation

SIq). AifIII1lc ~ arc charac:tcrizrd b)' l1li A !po Thr.(ity motif 10 thea actiyc

mt and by III ec-ad-ba .. c.uaJysil as lbcir mechanisms. oflCtioo. Cysteine proteasc:r.

-.. ~ hydrolym mtchlluun ID\Olvinll genaaI Kid·t.e formation foUowai by

~ly5JS of _ .. "I-tbiol iuk:t1"acdi.k. The a:li~ily of mclalJoprOle.ue\ depeDds

on the bJndtag of a div.Jc!tU metal ioa 10 • His-GJu-X_·X~His ..... if.

PnutiO ~ kno¥.-a to be ~ed by planb. animal$ and microbes.

Ho'4"C\'eT. m dUs study •• R"vi.:w on plw aat.l anirnal JOU~ III ouJ of "Cope and

the re-t;icw lS n:slfic;lc4 ont)o to mlcl"OOJ'g3llisms..

24

M1CI'Obcs have dra .... TI the attention as ideal source 01 pro.euet. and IiOOOUIIt

for approximately 40% of the loud worldwide t'fl1}'fM Ales.. M~

IqRSCnI aD excellent source of enzymes owing: 10 lhc:ir broad biothcmiQlI

dJ",""Y ....t Ibcir susceptibility 10 seoetK ICWlipulalita. lky R'p'C5mI _

al1lKtive souru of proItun .u they QI'I Ix cuImred III ... quaatitica lA •

rrlllJvely short time by esaabJisbcd fC11DClllatioa ltldbods • .ad they produce _

abuadaot, regular supply of Ibe desited procIuct In geucnL micrubW pr~ arc

eXU'lCrJJuJar in Dahft IDd 4ft din:cUy 5C'CmCd iaIo the fermmWjoo brvch by tk

producer, lhus ~imp1ifyin& dowmuam prooes.swg of the c:Ill)'me as cOCllplRd 10

proteues obtained (nu pIanh: and ..w..b.. They ha,"e a Ioogcf sklf life aDd cu

br S10rcd under less than ideal ooadn~ fur wa:h ..... ithout I.igfllficanl In ss "r 1Cti\;ty,

Dc::spi'e tbe long Ji'" ofrrotase-producing microorganisms. only a few are

con5ilkred as appropriote producc..-rs rOf commercial eKploitation, being

'gmelically regarded as safc' (GRAS), nOG-toxic and non-pathr.)gcnic, A large

aaunbu of microta belonpl to bacteria. fungi. y~<;t and acdnomycctes are

kDooA-n 10 produce .JbUne pmlea.!Ot!l of the toCnne type (Kumar and Takagi1 (999).

SGmc ofthc n:portcd proCCatlal arc Ij"cd in the TabJe 2.2.

25

'1'1"1. 1.Z OpUmll,d produl'doll eondltfons foralkaUne proleate produclnl mlcrnoru_nilml

M IClooflanl'M' pH Ttn1l1 ,I\~t~hvn 1n~"\Ib4ltlw PRol'm-cd l'tt(..,..dl Il."rm". (C) 1"",,1 pmM{'" IL"ptlmised Mrug~ gprimlHd ur""'"

~ 1f.IW\'eI ._-.. e.". ... k.l'lf'IIr, fo'"f"II/,t •. 0 J() 1110 .. ,. So}-beu .:.1 s-" n........ IIId ~ku""',lOOO 1I,. .. II" •• p 1S· J lO' J' 100 tZ ~meaf GIIKOMI ..... " ... 1.1 ... "111;11«, Jp 1f"9 10.0 " UO 24 NaNO, Cilri!: Kid ./Mllwsly .ncJ N.a, 2001 11.",11,.. JP 1(2 '0 ~7 ~~ 6\J 1J Caeinh~ CiI)'C'mK 'lamall et ,I. I""

~J.bmc. lord,.. qtAom .. H( HIlt \() '00 1~ Sua_.I .... 2~ 11~1l/aJ r~' IJII 'l 4flO "0 ("udll Cln .. h "~'I et el. XIIM IIH UIw lIP "·1 9,' )(1 24 ~.~~t'*'1 GIUII.'UM' 1(..,., el,. *t '11'41'1"19 1tOA·14 1,(1 )1 200 .t1-~ ~ 1RCIol; peptCMIe SIIIdI O~ et 1'.lOClI;

run .hl. JOUZ 1I"/'IIt.,, IIJ' !:I!,I.1t I HID 4t' I ,<I ta Bi~ H",mlM1. tKIaN IIIIP.t Ill" 204>1,

~It\Lrol(l

H, ~I'fl'/ .. M T('{: MOO If, HI' n 21)11 IX! ~meal U~'IUR IWIfrJ.a et .f" I ,~. S, Itt'hr .. ~I/'f"'t '1,0 ,\1/ l!U .. 4UII ,1111 ~. (NH.hl'O~ t..lJto ••. ¥l.,t;UIt Mllb,\1IU. fill" 1 '1111,1 ATe" 214', " "1"',III""IIJ" 1,0 SO ~OO HO 14 ('tiein at Cumnillll (HUCMO 1' .. " al-. lO02.

~

S, I'ftlPtIT'fI MK6 J U U- 1'U 60 (,~licpa.~ (JIIICDR. W...,.. 'Itnft ~rnM,)002 ',o\J'I~n,. n,.~ lO )00 nJl. D~ ~t:alJU.t OIIltCllll: 5."", lit 11 .• JAK' Ib , #tIN.h. J 6' .. ,~ ')6 250 , . NIItriad brudI; y.:uI ,~ Cil~. ,-l"1rICt _ .......... 1.("'

nlll'g~·". ' .. 10 ''0 II I'aIJIII:IIIOne,. ~ Ncme' '-t,.. • 61, 199. t.1 .. rt ....... PlO. QIr&t.~ l'-.rImatJUJ "'0 }1 Mtk 204-AI ......... t.2flm

1If"~" ~. -'>'111 ,. •• 3f," l.SO 14. J6 J' Ye.r atJMt.1I?;II'one. WlMy.NmIM .......... I1.200l; I\T(( 1,110 .~NJf.I:I ~et".l", ,Wm,r. ~ntl1U 1.6 J.O lOO •• ~"'.I..:ocM J·,brrtJ ~'r:;t 1J )0 7W '" ~."'II ... al. 2QOJ

, ... .• ~IlIU~ 1.0 " 2~O .4JI'fJ' ,/Uwl u~:n.,., U \n IIUl 12 ., IliSfJ HI 72 CQ/w1/lItY,Jluf I'£INlIIII'''' '1-H ,~ ~2U 4H AmmUltlum Ili~". Iryph ... ,. Sll~""" (Nn IU. ".20) e.seta tJplt/(1 ',mM rjlVltl ".~ 2J ml tlll.Iyw ~jdliok fi'o;lnl ~~II m~1 "l.rch r;/ti/..,,'Ir/U/IIlJJ/lI/IfI '.9 U 204) 24 120 I'eplODl. yust t=II:mt~l,

NaN01.INH.'~O~ fHIJcnlG

"clinllm)rttCl &~I~'ft'j Jp. '1 lit, I" 24 R.pancaJ NYot' NCIB 10070 T¥wIOIrM--:t'fn "', 7] '0 1 "Cl 1ft ~ SceIdt [79

A No c.rbiM. .0111'1£'" p, ..... , ," the ~ and ~ mlJIII' DtpmC Di-fnFn ~.uppImI a.- raprllltl ....... i>~1 ~I*idld

w., !If la . 2001-~"'''I/lII-. JaG!

111_" jft .1, l"~ AJ,"'.m and M",II11, 1~"fI

BKtcna MC the """~t dummant group at most commcnull ~.

-mfy nruual and albhn( rrot~ prodUL"l."tlt Vollb tbc gcaus BtK,lI ... ~ms tb.:

IIIDII pmnnnmt ~~ A mynad of &1nl,," ,...an front 1TIIftY dlffcrml Cllolic

cuvirunmrnl' M\oe been ur'um:l MId cxplmk'd for ,lk:;d_ prtlCl'1UC productioa

buI mo~t r-~tl.1 .1bl~ prOlt.AR pt'od"'Ul8 *ilb ~ RraIDI or B

1./w-.,/I)rJffI.t, 8 $Ubnlu. /l ~"''''dlupnf~t'"I",l. ePtd B ~JlIlrlUu (Gupta n .J.

2OQ2b~ lti.Ah"l. 1911; Kumar'" T.~ 1994. Rao et at .. 19Yk) AnochcrbKlcnal

SOIlf'CC. lllown .. s. potmlyl producJer. is Pr~/""'tI"4S \p. (Ra~ d 41.. 2000~

Ogiuo cl .tl. ! '"'J) The NctC'flaJ ncwraJ p!OlttUn m' charactrn~ hy ihe1r hlgb

affimty 1(-., h~dMpIk~b-t~ .... uno acid paJl'1 !belt 10'" thmnrltu~ l~

ad\oaularcuu, (ur conttulhn~ rhnr reac1.;\·u)' d.&1lft8 the prodU('hun "r food

hydrolysate's with .. Iow degree uf h)'droJ)1N~. Some ohbc MUln,1 pr01tUft belong

to the mt1D1I(lprllt~ase ryrc: IUtd rC'quJr~ dn.-alcnt ~lal ions fm tbeir Ik!ltviC~ ...... hiJr

och~ ate: ~cnrH: prOltln.1.\C!I. ",Iuch are DOt afJwrd b)' c:behIlJngltcCnli. 8acttnlJ

alkaline' rW1~IIIIC'li are chJltlu.1t'f1a:d by ~ir bJgh activity at alkaline pH. I',g. pH

10.0, "Id thci .. hruad ~b"lfa'e fop«ific'ry, Their optimal tCtnpc11lturc is around

604 C. Tbr)(' prupcon .... ' of Ndcri.! alkaline prlllUlICJl make them f,ul .. hl~ (or US(' in

IlK" .:ktrfgcnt l~ fhl1p ~""'"",'..oo""O~hHtk'..:h ,am)

futltJ ~lAblntc • WIde' unCI)' of CnzytDt'.lhan do bKk'na Fm ... umpl ....

. ~II"" ,.,,'="" prodlK"C'o .... :Id. neutral and a-b).ne" protctsn J·UftpJ C'1JZYIDC3'

QD be con\'cnimtl) pmdul'cd In =-~ fc-mxntation pr ... ~ The funpl

~ arr ~U\( 0'''''''' ~dt pH ~ ,.0 to 110, aDd ntub •• Mu.d ,ubstnk

sreafiru) Hn'A't'\ef. W) h.~-r • Jo..-~ ~ rat! .and \\'0fW hr ... k.k:,aocc thuJ

do dac t--,tTW ~ Ftm .... K1d ~ ha,,''''''' opt,"", pit bct-a:n 4.0

aDd .. 5 Md ~R" ,ubk hcn.L"n1 pH 2.!i .Id hn. FUUP) QC'Utm JI"'4~ ~ mcta1~t\>1tt.~s dw ar ... K'tl\"(, ~ pH ~.O and :In: InJubUed t.t) clad ... ne agtn'.s IEl

~" ·h' .... ' )illl. {(aaitrabam d "J • 2000; R&)AftWIIU IDd Hilcb. I QM7. s.dhy. tt

al. 200~. UoIM ntllll ,"'Ipfn.ccd group. md COlfJt/w/toIlU $po tBhositlc et Il .. lQ9~),

P81icilltUlfl tp (GmnaDO ~I al . 201H.lIIOd RIU:::I"".., Jp, fBlDerjec' and Bhattacharyya..

28

1993) produce protease. Among lICIinom)'CC16. mauts oC ~,ftS ~ the

JRkrml SOUIU (PdinMe et al .• 1999). Amaug yaasb. C41fl1JdD 'P- has been

5IUdic:d iD detail as a rotenfial alkaline proICaX produca (POll et al.. 2(01).

Vinl prot.casCS haYe pinrd ~ due to lbciT fUDCItOftDl involvement

ia Ihe proccss:in@ or pRMnD$ or wUuses that C3IISII: cmaia ratal dba&Q wch •

AIDS and cancct". Serine. atparbc and cysteine pcpeiducs ue f'0UDd la \'arious

virusn (bwliDgl aud &.m:a. 1993). All of the virus eDalded pqttidIMs Ire

~ cMre ~ DO IDdaRopcptida$e\. Retroviral aspanyl prokaSCS IbM

Ire required fOT \-i:ral assembly and rcplkalion me homodimcn and ~ elpt'CSKd

as a pan oftbc poIyprotein prttUBOr. nle mature protease is rdased by IlUloIysts

of tbe precursor-An ex tcnsive I j'erature i, .~ilabk on tbe expn:ssion .. purlftcalion.

aDd mz:ymalic analysis of rccroviral upantc protease ... lIS mulanu (Kuo and

Sh.rcr .• 1994). Extensive TI:!ieafch hft focused on the three.ciiJJ1C!nltional structure

of viral protease~ and their interaction with synthetic inhibiton wirh a view 10

dr.signing potcm inhihilon Illat can combat the relcnlJes51y !lprcading and

devaMtadng epidemic of AIDS lPatick and Pom. I 99K).

Tb&: nariDr: cnvironn.ent. "'hleh encompa$SCS aboul 71 pcrccm of tbe

Eanh·s surfKe. is polftIlially. Vd f'CWUI'CC fot bSC'fuJ en7.yI1lC5.. M~ Iwc In

tW1DUS babilaas m tbr marine C:lwironment.. including neusIOIl. pUnklon. ockIOIl.

rcsIoan MId ~ic. erutnbiMit.. pdaglC ;md bmrhic c:mUOhi'DC!Dts.. Thew hAbiuu.

Iwbor a divenc ran. of IDlcrubc1 including .rhachK1aa. C)"IDObIcla1a.

actiDomycclcs. yeasIS. filamentoua fungi. JIIH.'To.Ipc.. afpc 3Dd pr0t01Oll_ Atm..

alJ lbese groups an: JK*ntW SOUftU 0( IISdul ~ 1111: ecologtcal role or lhnc: ~ ~ ID tbe 11Dttiibatioa ead fU)'Cling of compI~x urpIllt

matter through degradalj~-e pMhways .... 11"15 abo contnbutc to the sec:ondlry

produc«ioo in the sea. Bacleria IDd futJgi 'leCR!le diffcrcot Cf1rynlei South aI

protease. amy~ bpasc. chi~~. «l1uJaie. lignina~. pet1ina::ic. xy~.

29

o.,,,,.l

cuclQ.\C't C ~ R. ~ua. R"s.&nc1lcXl aU:~:dIn ftc .• d(. baKd "" &Mu b3lKtII

aad ~.. hmcbom fbnb DanDc m\1fOMJC'n1,- such as &cp ocean.

byd:rc8cnnaJ vents. po .. «an5 lOll eatmneJy tabne bodo 01 - .. cr. have

yaddtd valW&blc: e.tttmophlh(' rnlcr~ ... hleb are ~ pnrrAry souru of

C1ll)1DrS that an: ar.;U"c: at el.UerT1C' ,onditioru Aft alabl~e re\1C'\Ao' of mann.c

nucrt'blll ml1>1nC'S is prt'K"Iltcd b) Chandra!.ck..van 4nd Kunw C :!OOl. Mannc

mkroblill (nI)'~s havt' lwcume lhl: focal POInI of mterest and ~vtrat enzyme.

hue dr-swn the attention 01' nlil:rohiaJ prospectors. A rew &!"lyltl~i ha\'C! been

lllQlated rrom ~a""'lltcr am) n\lnOC: scdilHc:t1b and have been punfied and

c~tnucJ for Wit propcrttC\ and apphahom.

IlK nwUt(' JJlrCrol~ .. lricb ,.'u· ~ pttftcftMI '*' .MWU of

nom...~mc .. ~ )d to bt &.aprt'd rn ip~ of fc-,r. rcpon.t ~ the- productiC'lll uf

PII"'M~ rnl2Tl marine b..1cn..t ~ftn Ilk Vr/mfl 'p. SGnlillS '" _ PY'WlOfflUIWJ

.. eh: ({'rood;cr et 1.1.. 19Q'J~ fJo1l..-ia-Bad.Uo n •• 2OOJ. F.~' and Crosa. 1991.

KUITD&f d.1 • ~. Makino (1<1'. IQ81. SaQmanc<l C'C '1. 20(2) "C',,~kn. lbr

produ.;t, •• ur prCle.lSC At ;&n IndustrW scale using mIflM funp~ I~ ~. lO ~

Incmpt(d IChand.ra.~k..uan and Kurrw-.l00:)

A halolOIn-etlt Iftllin ur 8acillus 'iC'h~niffl""'ll, if-olaled from mal'inr

IJOCI.itoohb produced bigh protCUM: lictiviry dunng the early !itationlal')' pnasc of

powth t~lltllK'hinl and fontn-. (998). Th( UlIC' uf SoC. \l.~ICr 10 1he fcnncntatlon

medJum cnhanced the: proc.t&ktlOO of this KtJ\'lty to I ~O% "iter pU1I.l

... r,ulloo_ lhr« diffemu pu.rol)'Uc cozymn ,;ould be: de\c(,rd v.il1<:h WCtr

aIbImc Ic:nnr proIca5es. c,hjhihoa opI.UNIl kllvily .. pH 9_0 IDd at: 7Cr'{.

Procca.~~ "ttc .ro\~ by '.1...-1. .ith a ahrc:r- told Incrca5c In IICh\ilty met weft

Albk In tbC' ~ <'f 0 ~~. ,altO .. 0..5-. s.t -K) aDd 3 .... HP:

Ir1lf1t,U]luJar prOIlC'lltSn rnrrn • h}'peI' Ibmnoph.ilic III"ChaNn P)'rtX« ....

~" W1IIIC punflCd b)' Halia et aI .1991) ~ Pfpl has • m .. l«alar mass of

30

19kDa aod 0fXtIB in at least two c:oa.fOl'mllioaaJ ~ j..c~ one as 8 ~ "'l'" aaolecular mau 124.16 k.O& COIDpI'WnI about 90% of the tobIldivily .ad the

KaJI'Id one occurring as, • tnmcr ,,-nh 8 mo~uJar mas.s of $9:J kDL Tbc:

kuapemure optimum ..... Ibr oormaJ Pfpl .. - 85"'C whete as... chc: enzymr

pftpiIfcd by iacutatias me ull ~:mKt of P . .forionts III ncc in 1 ~ SOS fur 24b..

-.1000('. Morcovtt~ dIr halflife was iDnTascd from less Ihan JO minuln 10 31h.

The CftZ)'mIe appeas to be 8 prcdocndaIal Jainr-l)'pe protcuc m «11 c:-.trKl. bu& a eoa~la "..,tro. probably 1ft put. byclalmiddicm of~Am' aDd °GIn' rn~ eo

• more thermally Mablc funn by prokxtged heat tra.tmaJL

A hypcrthermophllic and barophilic protease \\iU i~latal from

Mt'lhlJ'IOCOCCUS j(Ul1lQsdlt. an extremely thermophilic deep-sea mcthanngm. ThiJ

mzymc .~ lhe first protca60 isol.ted from aD argani5D'l adapted 10 I rush prtnun:

and high temperature environment The: enzyme hili a molecular RtIW of 29kDa

and narrow substrate specif'icity with litrong preference for leucine at Ibe PI site of

polypeptide substrates. Enzyme ftl,.ltivily wa.q measured up to 130GC. EtU:yme

.clivil), and thennal 51abiliry intrc.alOd with presMIfC. Rai~ in the pressure La 500

atm. led 10 iDm:a~ reaction rate If 12~·C to 3.4 fold and (he IhennolUbibty to

2.7 fold P.fichels and Clark. 1997).

An alkaline: ~ isolated &om • symhiotic: baClaium found in Ihc gland

or BlMrine shi(N.um was ~VaIUatCd as a clczm1ng addilive. The addition of thi$

pro1CaSe signifiamt1y iqJnMd the cleaning powa of ~c dctetgtnl£. The:

proIaIC was SUIbk iD sodium pertxak. • MD as ~n peroxide lad maincd

good actr.-"Y in the prescoce 0( tOCIium h)-pochJuride (Grcme et at ... 1996l.

T-v a01l ccllubr proIeaSe 6um c.rude oil degndj.os marine .NoautIiopsu

•• isolaled. (Dixi1 aad haI. 20(0). The two pRMeaSCS. Protease IInd Plnceacoe U.

belonging to alkaJine ~ mdopcplida$c ranuty. ba,,1: a mokcuIu weighl uf 21

kDllad 23 Ula and PI 0( 8.3 and 7, with pH IIId kn.perarure oplil1ll (\.'If adiloit)"

bt1wren 10.0 and 110 ~nd .00'" wile respcc.tively.

31

A S fokt U1Cl'Q:K in ~ prodacbon I1'IInIrIbe badtnum , ,/tno Cn~

~. the .cIdltJOn of skuruned milk ,,1UdJ has a molecular .'ftIIu of J.lkDa has beca

~N c E.s:a.ta-a.dtUo et at. 2oo},.

An OllldatWC _ §DS uabk aJbli:nt proIfti~ .-as rrponcd 1(1 be sumed

by mmnc Mlophdic 8".,/lwl .-I1IlUU, ~1arcd from w tHSa.J mud n.t\ of Kottaa

Yello".. Sc-.1 4KLlmlr et al. ~000I) TIle ~ ~ho"ed C'\I~mc \.Sahlbt, ""'m1~

SOS and olldWaa apDtl. rn.H:ag il5 X'livity .htwc % .nd 7~~ Oh lleabDtal for

72 hn ..-irh ~~ .. SOS lad ~ ... H;O; The cntymt btlonp to thr family ~

prottaiC \\-.lh ,,,,lIma1 pH and t, ... Ptllfti!~ of JlctJ\"11Y'1 11-~ and Im-C rnp«U\'C'y

Z.S "" .. Me Production lD M'c'DOI'I •• iI ..

Pru4~ prociuc:titm l" Itn inberlml property of all orpnjtotnl and thChC

mzymn .,~ gcnnaUy constitutive: howe~r. 41 t1tllCS. whC'), an pan •• J1)' )nduclble

(Beg et al T ~OO1a: Kahn .. l'ItcH Protases arc: largeJy 5eCfelcd Llunn" \tarioruary

phase Md thUl are JC'nchJl~ ~gul.ted b)' carbun and nitrogen "'Tt"'«, They ~

Ic.nown to be a.\MlClatcd Vlnh the unset of staliooary phuC', which is mark~ by &bC'

tnmsltlOD iTom \'~lCtaliYC fIJt'\\1b to ~)On uage in spore-fOf1TU!b ll1erefcn.

peotaY' rn~"'U,"CiOD u. oOcn tru.ltd t--.) the. srcwrulauon ,,. ID man~' bacilli. such b

B_ ~/""u .01 fltll and tfaBC'man.. 1990) .od B I"'k,,.!onrw I Hmtun IIIJd H~.

1981) In ~ontru&. la f~w ~ al~ wage.1 IbM spoNlalloo and prolCIIK

proclucttM, iJIhouP co-occumng. 11ft DOl "laled. as lopCft-def.oa.f sttaias of B.

ll('''''''f~u .. ~ noI ~ dri'H:imt «. knun, Cl al ~ 1 ~ I It ___ .00

esubl • .ba! IhII rrocea.w prodllCtk'lft Md )porulAllot1 MC' ""'0 cradcrmdnlt n~ In

5Wlon&ry Jlh.'l-ie by aaaJ,.·-'&c 01 nudeoIade poo~ (GTP and "T f') III IJx cd! or;

(Bierbaum d al .• J9Q1l llww ~.ioru. mongJy MJU."SI , ... pn1IQ5t

JIfOdu.,."1IOft I1 un&r ilNlfml c:onuol rnpons.~C" to ;uNDO acid dtfiC"lnJCy aod l§

~ Co ~ Gppp ~lIo tft the ceU The 1l'aMlbOIlS ~cen d1ffC'ft'IlI p",tla

~ VC" d,ff(,mlt nutnuonaJ hmilanOBS v.tr~ ~Iy dnccmcd It)' IlK- .. lrtntimu in

Ibr DUClcv .... pool. A nwk.t'LI ~fcast in , .... GIP COIIIt'fIt or IlK- cells (aftcf

32

Idditioa of mycophenolic acid in the apoacmlial phase) inaa5cd protease

producIioa during mUODII')' pe..e. tIenee 9 it is conduJi~ty ~ dw

abCdJuJar protase prodItcta ... • mmifeStalion of aUlricnl timiabon •• the

ansa of SlMfoaIry pha5c (BXfbaum ~ al .• J 9" I).

1.6 Ftt-. .................. ofprotaM

~ an: FDl!JIDy prodDced by submerged fcnnmtatlon (Smf)'" on •

CUllllDeJcial scale. this is pdaied ~ solid sD1e rmneot3boa (SSF). Rtaady SSF

unpmcCS5Cd or mode.atety proc;:nKd I1IW materials. funbcr. the less initial tapilal

COS1. superior prcducIavity. low wafer ourput and impmvfli product ~ ~

Olhu ~ of SSF, Diffcrmc methods in submerged and solid itate

fmncntations have been u.~ to rqulalC protease s)'Qlb~ts with mategie5

combining fcd·batch. ('.Qntinuous. chcmM1:at cultures etc. (Gupta et -I.. 2002b;

Hameed et al. 1999; Sandhya c1 al .• 2005~ Uyar and BaysaL 20(4). Such Iltrlltcgic5

can achieve high yields of alkaline protease in tbe fcnncntation medium over "

longer period of iDcubal:KMl during prolooged stationary stale. Since the final pmltue

yieJd during this phase i9 alllO determined by the hioma.o;;s produaxt duri"B

tXpoIICD.IiaJ phase, medium manipulation is needed at maximize grov.1b ad beace

proIaSC yi~ld. Optimi.z;alico etf Ihe medium 1S associated wilb • Iwpc number or JIb!.'Iiological and nulnltoaaI ~ lha! effect pruIcatc produdion. ViZ.. pH.

1Cmpn3IUrC.. inaJbeIida period and aglbdtCJIL clfca of arboa Md a.i1JVFn and

dJ"*nI catiam.. AIIhough • 1arJe army of facIots inftueDces proecase production,

tIII:R lS • compIa ude:actlou amona chcsc ~ IbaI am be iIUdIaI by

followiag lespoIiSC surface mctbods.. A cowprdacnsM acaJUDI or cuIbft coadInonI.

for proaease pnxIuctian fron , .... rmcroorpab-ms is liDed in TJIbk 2.2

33

Solid ,,'41.: frnnt'l'lt.:ut\)n ~f()('r:S~~!i, wh;c:h in\uh:~ the: Mtowth of

rni-."tOOf1taJlism on rnotir ~tid ,ub!lcnarcs. in the .Ibtcncc or &tt. nowins Wltrry have

cOCludcnblr economu:~) potential ,0 produu., prl~~ for the food. feed.

~ntticak .ad aarinlt ..... ID&tusIries. fIowna'. owin& &0 Ik JI'etI t-UCUSS of

*F"'Kalc ~,cd ft'r.'nCfl .... i • ..., _Smrll"'""r'~. S5f ... almos1 (l",~

tqi«trd ~. 0YCI'the: pi 1>'20 ~ SSF has. ~ raac:wed RJICreII

hcau.ic " has cC11am ad\'antage1 0\ er 5mf (Barriot·C.onuJcl and MCJ11. 1996),

$,SF technology hit'" been Uo~l1 r.n rrnturi~1't frem Dpproxlmlltdy 2600

~,( 1* "4i u..~ hy the Egyptian., t\.lf making hread. ~d information on the "Koji

prexns" dnlc§ tMrk lO 1&)00 8(' SSF pton:'~~ Ilwt ha~e c~u.lcd for .. :enlld111

IDC:udc fcnnaul'd r~, r g _ latl('dt. IDlSO ~ p()lul). mold n('Cued (bene (e-,.

ROIIUCrOr1~. SbtIn ,uJauto,. for (('1"md)tN bmIo"J and c:mllmc .nd cornposmJ

~ m.-cm DPtM.""4JIh 0( SSf Itkluak Ihc proc.nn ~ clf apt .. lftdustriIl

,n.;d~~, the production uf mzymn. orprnc aod.5 aDd ((ha .unp: mcuboJlIII

.IDt spore" prOOu..·lI~b (ltlrtnNuJl, I WS),

At. part ur the ,;catch for I cheap alternallve produc1ion ~>'fotr:ln which

utifiu~ ~lmplcr.~ lDdmtnal W.,ln *' Jubmaca. solid.wc iermeutaUOD (SSf)

has p.-ne.i Impon.aIKl! ID thl! pMdtlrtK1l1 af [lIcrob •• l CUl)'IntS. SSf }w. s.evaaI

«"ODCJmK .Wqft owr oonvcnltoGll SUbnlCp fmDealalKJIII sudI ~ mini,...

~~. of ",&lCr. ~ uf lDCtIbob1a iD • ~ ~ fOfDl ..

11IIl.ana !he ~ procaslac ~ ttUE Q..If1)11111t\~ ...s ac- n,..,~

(Heuekmt. 1.,7:, ~ et 11 .• 193~) Among 1be uno,,' groups 01

mecroorpnu,,,,, uaed ia SSF. fiJ.mrntom fWtgi Me me IIIiJM wtdtl)' "ploited

ov,ing tt' thtir abaht)' 10 gf(l" &Dd J'foducc • ",dc: range of ncnw:cllular l'tlZyInCI

uru:mnpln 50Jjd lI"Mtnnt~ tMoo.yoonSICt 31.. I98J ,.

In SSJo. JUM,:tuuq&UI~ po- l!I1 Ibc motS1 .obd sUJ'rl""'. nthcT ,-.a mal

~n Of 011 ihMltublc subs.traln lbal can, III lddulOO., be U"iicd • carboo ..t

energy source_ The fermentation takes place in Ihe absmee UT neat absence CIf free

WIlIer. chus being close 10 the natural environment to which mkroorganisms are

adapted Frtt water does not appear .0 be tb~ natural milieu for t.hc: majority of

microorgm~ ~ot a'ftl marine mi~ prefer swimming in free

5e8\\"3ltt since more lhao 98~~ of isolal~ from the marine enviromnan ho\"C' b«n

obcained ftom the underwater surfaco of solid substRlCS., and less than I ~ of all

knOVr'n fungi have heen found in marine habdats (Kelecom. ~OO2). The cvoJutioo

of higher fungi took place on solid growth substrates. Ascomycetes ilOd

BlHjdiomyrer.es sopent their evolutionary bistory as 1cm:5Iriil!s. with onJy some

species adapling 10 ",-aler. later in their cvotUtiOIL fungaJ producb of

biotechnologicaJ interest. i.c .• t1lZ)mes, secondary metaboiiees and spores. wen:

developed for use in mois[ solid !'ruhstrales but not in tiqwds. Conscqucnlly. the

cultivatillD of ml('7uorganisms in u-tlucou~ suspension may rather impair tootr metabolic efficiency. Tn this respect, submerged fermentation tecbnology (SmF)

may be considered as a kind of violalion of the natural habirats of wild·type

microcuganl!>l1lS_ However. 1\olid state (suhstrate) fermentatIOn (SSF) is currently

u~ed only to a ~mall extent for enzyme and sccondary metabnlitc produclion

because of severe process engineering pJ'Oblems.

Approximately 90% of all industrial I!ozymcs an: produC4.'CI in Smf"

frequent]}' using specirlCaUy op.irnil~d. genetically manipulated lUicroorgarusm.~,

In this respect Smf procasiag offers aD insunnounlabk advBnlllb'C m"a- SSF. On

the other band. almost all these CIlZ)'11lCS could also be produced in SSF using Ytild·

type microorganisms (H~Jktf et ~". 20(4). IntCRStingly. fun~ yast:. and Nl"kna

lhal \IrCre tcSllcd in SSF in roccnl ~ cxhi1:Hted different melabohc Sltaleg.c:&

under conditions. of §Obd sfak and ~ubmergccl lamentation. AdWDla~ or SSF

over SmF are listed in the Table 2.3.

35

r ... u ... ec ...... ~ ..... oI ..... st .. ra. ... ,.,.. (SSF)

..... 't .01llft'J'" fft'1BCefadN ts.J)

.w.. .... .............. u.. -111ft 4UI&ld

Utp ~ oftk ctrd lWo4K'I

~;1r ~ .LJDItiua:dI~ lawn Of rntml"

(;1IhA.a ... -1 .......... 11*

So~d M.lPpuft f", mJ""'PI'1lUt1 SimW4tallCl .!(,Iw n.n,,'. ~\ uonmna'

Fcrmenbniul1 l,r Mtcf·tnsah.bk !/Uhd .Ilhmak:s Mi~td &:ulhlftl or nU~toUrv..nI"m.

P'nIIcHiI ... ch' ....... Hlgb.\\1 lunlC' rroducti\'11)

Lo ... mal)' *'rna.nd (or he.laflna tltili ... fin" ", "~I.tw ... WIII.llblc a.rboa IiOUI'tt.

Nu ami· IC'l.U1 ,hnnlcalJ,

f~illdw

prr:aacc d &M'ow Ihab t;UIIL"CldDlann or Ibr ~~

;t.tn.c-d cult..ftt of frnnmnnr llllauorpci ....

S<11C't pcd~~ !H .. -yJu .. ~

mlC'rgorpnivmc

Synnaism 0( mc18buh" rcrfarmmrc

Slballer fenntnk, \utUInC.'II

Euy IlCtIUDft

nlC'ap and abID.I'''' &:IIlrh.," MtUr'..lt

\:0108 of mU"~ dimISI trt'l'nrnu.uo:

--------------~ -----------~-----

11 ha, ll'O b«orm d(ar thallhe cost-factor for W prodak"flun of ""bu&­

tqR- CQ1)'IQL"S an moJI c.c-s ran'll&n SSF O\'U SmF T~ C J9%J. cpu_cd

fmneuullOn (Bob pf c~JM .. ~ rrudu.:1ion .1 CS $ 02 kg·' an .. la-SlCU SSF. ill

~ 10 l'S S:O ~ I m I ~t~ tad ~ t)nr tmpttn..nt b);)k~J fKlOf in

fa ... -aw of S.~F 14.~ ,!wo t" •. -.. ~)lltl' R!pI"C'UIOII. YlhKh AfPCarc-S It, tWl: hmiung

CllZ}mt JIfOdUll11Ofl b)' '~'XIII1I.1 "'V' in Smf (N."y,kuww et .. • 1(99), ~

lack of ~.ltk ,~tOfl 011 •• :.JSowcd for (AI JfO"1b of lJIco funps in the

p1~c ot hl~1 'upr (OIKCfllrahon1o ~ ... ea·T..mt"lo rt al. I~SJ

Sc"cral fun" na.,J I w.hd wt-mau ti oUI ~ for opUfniel p~'th MId

prodlK'fwdy T'I..:n:-fo"_ !C:II~hQ,lI, Ultodific:d organ ... ms.. IA'hleh w~r~ uptlmased for

RnIftI' "/lil,.,.,..,.

liquid ndti\'alion conditions. have often been used in Smf'_ W~ ulUR1

dollIeS ha\"e played a JDljor role in SSF,

AftOther probtem ..... could IM.'II be satisfKsorily -r"ftl in Smf is the

cbanw: in oxygen aouppty _ 10 C'han&ts ID tfx .,.,th mcdJum duriaa the COUDc

of fcrtnaatioD. lA many oucs- fimIi accd • highJ)' \'i$cous medium rot S«teIion

or the nquimi mctIboIitts. Thjs "iK'Mily is ac~td thn::Iu8h ICCmioa of

polymeric JuhAlDCCS durins funpJ growtIt In such cases. SSF IIpJKWS to be the

bcucr allcma!ive since slJrrer speed and OXygcrI supply play no role (Elibol and

Mu\-~ 1991).

Although a direct comparison between SSF and SmF ii. very dIfficult dH

10 the differem: c.onsisteneics of lhc microbial cultures U5Cd in tIu: two l«hnolnglt'S"

microorganisms involved in SSf' hive I higher metabolic potentjal .inee they

proJlferate in an almost natuml environmcnt, i.e .• under conditions of limited f~c

wnter and with a solid support (or BJUwlb. Wh~rea5. there has been significllnt

development in SSF proc~ing. regarding bolh biochemical engineering and

rc1.K!tor design with tbe goal of Jelling up the prooess. very littl~ if any work ba,

been done. as yd to elucidale the """'ecul., and physiological background of Ibe

dirrerent behnior of indho)duaJ miaoorpnisms .. hUl cultn.nC'd on sobtb or ID

liqujd5. The pb~ologic:.J Iftd molecuw biologiad .spech. of microhW culliwtioa

CIa lbw. be reprded AS the c:um:nt "blKk box" of SSF biOlechnoJot:y (k:8i~bekc

er at. 2(02). Tb.is lnIy. md "'ill. he ~ by more focused comidmatioft of the

biofogjcaJ pma .. cters appIicllblc 10 SSf and SmF. 1Du.J. the pm.pm.f\'C IbM SSF

wl11 gain pre\uiDg ilgni(xaau ia the Industrial produaiGn or au)111Q. ~

mclabolires. and spores. ~ ... dd typ: mJaoorpoi:snli is wat1'UIed StftOe, ~

with SmF~ it u rJlf.ft cfl«li~ in tnen.l aspeclS iochad.pg lower merl)' and

sterility demands u welJ .. htchcr ...trilily of products md \'ariabihty of

microorpaisrm UKd.. ~illly IX use of mixed cullun:s.

37

Two typei or SSf s.~ c.l be cmtiDpisbcd dcpcDdjaa 011 t~ naaure of

Ihc sdid phase used ,. first. and the maa axnmoaty &lied S)'*'m inwoh'n

cohn'3lioo OD a DIIuraI mItmal. ~ secaad 'ys1aD. wtUdt is DOt .. ficqueDtJy

used. m\dves cubivlIbDa on an men wppon unpreaDIUd .,lh • I", medium

(8arJios..Goftza~ and Mrj~ 1996).

SSF cultinrioa an n.hnJ suIKuaIes uses IWUnI malarak Ihac 3Dft boIh

1'5 support and • nutrient SOUfCe. These materials ue l)'pical1y stan:h or

Jignoccnulosc baKd agncullural products or agro-iDdmcrial SOUKCS such as grains

and grain byproducts like cuuva. rotMo. supr ka pulp. beam. ~ Inn and

rice bran. The IDlid sUrP0rt or the sc:cond type 'Delude incrc mIICriaJs -.hich Kn.u

only 8.5 an anchor point rOT abe microorganism. MutcriaJ5 proposed include: hemp.

pcrlite, polyurethane foam (PUF) and vennicuJare (Barrios·OoD1..alez and Mejia,

1996: Larmchc and Gros. 1992; Larrocbe and Gras. 1997). lbc materials used in

SSF arc listed in the Table 2.4,

Tab" 2.4 SolIM' uampJfS or substrates and applkattoDs of SSF

SUMmit/support

38

Appllc:aUoDs

PrucC'dl earicbmmt LIPin dcJnd-tiaa f« .......... I'eechIock ~adtr.aoa

~pmda"'-Pr ...... ptOiJIactioa

f"VCQ~" l..Q ... ·wc Protein cariduta2ll Pca:iaJ1ia ........... L-bctX 8cid p~ ~ awdlOelll fat ICed hnlMk~ C dI'K .od pudatoc.

Vicnturs et al., 1981 Zadruil. 19112

CalDct. Lot 11 .• 1911 Sihun.191O Vil&cpi d .t .• 199) ~~a1. tCJ97 s.bu 1:1 al. 2000 C ........ ·aa.co et .... 1490 ~1!I ..... ftI S«.cuI et". 1994b ()urud tot I_ . I'llX

8acI;u: Cl ... 1994 ~_l..oMuc.

19'M So,ua c1 ... 2OUO

Sp .... ..,.". ~ro.. Po~1\'d

AnlnII produtriaa

~rm""'" ...-~ GfowdI ....... n.w- pmdacI_

fDpI~~ ~hd~ r.c.e. .. L .... for fad Amyt.rpr~

~~ (,ifOWIb~

t...p.Ic prududioa Gt~ Kid prudur .... l.(i~

C'h,.blUC

Y..adri Cl al .• _. ~ .... N1ktIc1l1994 Jtow:aMia rt ... 2000 Soaol Cl at ... J",,­Bramanb • al. 1991; 0rIIIaI Cl &I • lOOO

t...rrack et Ill... I'" H. huei8:,...t r,... 1993 IWd -"'."CI" • .t. 1.)

~-ChR Jraod ..... 1996

n. a al. I"'" AIaiII Cl .. ~ 1990 Cbri5Iaa 1:1"_. lW' Gdmi et aI_ 2000 Prabhuarut ~karut. 1'N6

St.arMMd ('handra!iCkanm. t 998

Ciln.. FCI OrO\lrLiUllldlH Leon ch!, 19U "",k pomlCe: [lJumol productIon NgIIdi Imd C()rrr:ia., 1992 Kiwlfnlil reel Cicri~ .cid pl\ldlKllon Hans lit Ill.. 19R7 _An_IIIfII_n_Ll ... ' ... ~ia ____ Vo_S_"_Ue_w_mpowd produ_t-,-Clon ___ I.I_mn_o_l1-,-k_i C!_I_Al-...:. •• _19.;,...9_" __

A ooasiderIbJe amoun1 of -'OI'k has been done in rcunl ~ to

undenQnd the biochemical _ engineering tipU1S of SS.' processing (Mitchcl1 et

.... 2OOOa; "Mcbtll et aJ .. 1000b) .• & lJ t1II.bcr liutpri.;ng lbat lhe lechnlcal pmbl~m50

of SSF ba,,-e DOC yd been solwd as SSf is ODe of lbe ol~ biuea:ru.o •• 1

~ knowm. A co~ of Ik ICador syItnnS u:sed iD SSf i, listed in &he

Table 2.S.

39

Tab1e 2. S Main rtat:torl uled In SS.' -

Ad".n ... ..,dna" hHU-Rtnhtr 8rl,( d~rlDUon I R.rd'!~" HC'I~'~ irlll., rrf'''''CJ''~ I

1:.l1l:'~auk flulL .topped ~tJ, conon wool 01' I Very _le. 1_ -. .... ......,l 01_ (Cbrta. et a4 IlJ9n AA"'tlllytB numerous Nn5InD rcguIarion. palM

IICI'1Ibun Pacbd btd tt1lC1or Colllmn mct(lr With Itr 1.0'" cost. CM)' RplabOft pr 1I:ftIFCnnft (F.1dI Cl 11.. lW6)

prcbumidiJklljOD and airflow..-. pmba ~~ (R""""'i It al., 1998) bwmDO\-al (Silman cult 1919t

(OunInd Cl DJ •• 1996~ Raambcwh and Aln7Al'l'l. 191Ul)

Pi/tI' piu", cmJ lu,'P .'ra/tI rttt.lUOf,¥

Trny tel(fOI r.rfonltC'd "Il«:l '" " nlQbcl~ni ... Good .endon. 110 beat bund "plna film" •• (Fuldl ct al,. 1991'1) "umber. covered wn.1I alhm layer or Jabor iDleDJive (Ohncf)lal et lit. , 9M') nbtulle [Dun,net cuI.. t 9961

(Daub, ... tC AI .• 1987) (ZIdruU tU!. 1996)

l)rum reactor IfOMNfttat .... indinc.<d &:)lialdc:aJ Pogibie aseptic c.oncluioM. ,,'nll.: Inbma. (H. aacI AadftJOD.

drum. AaJ!I2ion by • 1UIali ... ~em possibk uomuiutioftJ c:h.'''lftiflj ut iJlc 19751 (HeMehjno. 1977) • rouud W CltWnJ ui. III by ...... cs solid sub5cnte. poor heal removal . (LohlW fl al., 19", Vlilluo tbc! cocniDtr

fhJidUcdbtd Column 1'1:.:1.of with I ptrforattd base F.x:ccUc:nlllCfttiua, dfeclivr remov.l of IHona It Ill,. J IlKlO reactor 12110. which IIlr" blown with (or« mctIIbolic bcallltigh Wll

10 ""pad the ,ublht.: plntdes ~

OiA lermentor

17, .ymotJ"

Semi ..... 1OOWCd R,,-ulOr (Crow lbc lJni\'mCil)' ",f Santiallo, <:hik)

MOIt tlabonflld "'~l"IiCl'n af 1& Jdlm:d link ~D':IOl (workinA Y(1hunc -3.6"' ...... l:~ulf1f1'''cl with an ImellflS scmv for miNI""

Cyllndrl.:.al devJct wirh dilb ft.cd cm • rot.llh,'C hoolAlnUlJ WI. tlcb disk co..uc. m 1 -1«1 frieS. mlIOCainiD,g a l)'otbt1k support 1ICt4k.t with I matnli~ toIutloa

Cubtc rac:IDf with tn:., nchaater tbcttI (with water n: .. .,&:II".)

PI"~"'d bod ltKtor with pcri41C1k: !llium.,n. ~ophll1iClted COIlIToI of "mpcratl.lf':. w.&cr COfttel1f. bed A.lultlon

, Accurate coo,",, ohirfTu"N. hurnidily IfJd 1CInpef8twe. good mblinJ'hlsh In\'CIln~" and nllltnlonaUCC COlts, economieal1y cff'cct;vC) o"ly rot hiib added value proctllctJ

Good bomoawzation of the medium In" heat tnInIIfcr. dTlXlive (Of ~ productioni' lwd to ~Ic up. Ih:UY prod.,r recowry

Good Iat JQ1O\'aJ aDd .'nlfion. l1li)'

sWiog upino stirrina

D)oreactor opcnnlDn anulidmbly aiutplifitod. dticiem COTIcrol u( ~()' raramalc:rslcx~nsm !ut bulld'n.

(Dwaad lad Cb. 'm\l, 1918) (Dlllltld "al.. I"])

(Ralmbauh and P.oustoJ. 'V .. ~) (RUUJM41. clIl.. I ~ 1 )

cRau.ut et al •• 1993) C~ ••• ~"t .1.191')

"aDMdn et.t. ,WIt) (Femaadrz.t at .. It.n~

2.7 Prole •• PurifiulJoa MC'tbocb

Purifte .. jon o(protase. 10 bomogmeiIy is a paClcqWsiu: for Qudying dJeir

mechlaiim 0( lC1ioft" Vau n~ of puriflC8lion procedures for proIeases..

iO\lOJ\ling affirury duvmMognpby. iua-Gc=Imnsr ~". gel fihruioo

Icdmiqun and prepuali~ Jdyaaybmide !Cl rlrc:tropbornG ~ been I6ed r ... the puriftalion.

A aumber of albbnt pmteav:s &om differmt 5OUr'C:es biwe beal purified

and ctwxtulled and .. a.ummary of \'arioo.s purifJCalion ~tralqies ~ for

puriration ormiaob.al prokues l'l presented in Table 2,6. Tbm: 1ft DO set rulcs

for the punficanon of pmteJlU5. After St'paraling the culture from the fe:rmentation

broth by mua.ion or cL~trirugarjon. the culture supematIDl ii coDCI:ntratcd by

means of ultrlfitltation (Kang ~I al.. 1999; Smacctli et al.. 1999). saJtiog out by

solid ammonium I\ulr.lc (fluladilok·Towatana et 8L 1999; Kumar. 2002). or

solvenl extraction methods u$ing Ketooe (K.umar KDd THbgi. J 999; Thangam and

Rijkumar. 2(02) and rthW'Jol (EJ-Sbanshowy et al.. 1995). In addifi~ olba

~ web 8i use or PEG-lS.OOO (Uu-cher et al. 1996). KtI\'a1cd chamJaI

(Aibl el :tI .• 20(1). 1~~5C1Wti\'e bydJ.l (Han et -I.. 1995). Ma

ttatment of enxyme (Rahman Cl .1 ~ 1994) and lyophili1aCioo (MaoonmIRi and

Joseph. 19tH) arr .1., UKd for COIJCadraIioa of alkaline pR1CCI5n.

To funhet purify lhe cmyme. a comhiuatioa of oae or more ltdmiqucs 5

applied. .. iz.~ atr ... ry ~ (Aa. ion excbanF chromIlogYapby (lEC).

~c j~ chn::a.tograpby (HIC) BDd geJ fiJ.Iratim cbromatopaphy.

0IbcJ metbodJ 0( cboft. nx:1udiDJ aqurous ~ sysInDS (SiDbA cl ~

J996). dye lipnd duom11Ol:flpby lCowan and DIaiel. 1996) and rc.m frwtJoalUoa (BIaa}cc d Id .• 1993). baR also been employed on a small sc:alr aod.

}'CI .. a.it commerc.al e-rkHtalMm,

42

The IJ10St commoa affinity adsoItIcnls used for ...... proeeaa 111:

~Xyapaln.e (KobIIyuhi et .. L 1996). i1mIIcalilal N·~)QrbonyI

phcDylaIaDinr aprase (Un:hcr Cl .... 1996). in ..... +iJizaI anfta ~ Ked

(Manonmani and JaICph.. 1993). lpfaunin-aprtJK (PctiIIIIrc et L 1999) and QIaII .

..... (Humdilok· fO'4nl&na et eI_ 1999) .• o\lIbouP AC is ODe or die aIOS1 i4K"CC'SSful

purifJadun k:Cbniqaa.. • ..;or _1MiOa iltk high COIl of CIUynE HppOIU _ ck

tablk nature of the af1W1y tipndl.. whidllowm dIcir II5C ~ ~ .c:aIr.

1.7.11 .. ne ..... c ...... , ........ J (1t:C)

1bt: mMriccs for la:.c CCJDtalft ior.ailab&e ftmctional groups such •• dl(!chyl

amino ethyl (D£AE) and carbo2y methyl (CM). which get associa1ed with cbe clwJc:d

rroccsn molecule$. thf:rrby adsorbiaglbe proIe1n 10 dlc- malriccs. The adsorbed protein

molecule ~ eluted by • andianl change: in cbe pH or ionic strength uf the du\fna bLlffer

of501urion

2.7 .. 1 HydropJtobk latmwUoD "hrom.tOlr.pby (Hl(l

HJC exploits tM variability ef cxtmutl hydrophobic amino acid residues bn

difTc:n:nc prulcins. leading ttl rrocetn inwActJon by \i!tue of the facl that in aquooUJ

solvC"DlS. h)'dropbob~ patches on proteins prtrercnlia.Uy seek 001 odw2" hydrophl1bJe

~ Tbc:se bydropbobk iDlmlctit.- an: .. 'TaljJlbc:ned by high salt ~

I1Id higher tcmperatuta and Art wca1eoed by £be pra.cncc of dc:Ic1Jcnu. or miscible

orpnk sol\-mts. The QDI of bin~nl of. hydIOfIbobit protein dqJaIch m &be I)"e

ad demity of sub6t.isuUoa of dx .... tnx ... wcll u u. the UbaI'e of buffer ~.

H)'druphobic ~ are muc:h mote \"Iriabfc ln bchIvIor dla tOn c:.ct..,as alii.

because ofdus.. TCSOIutiun D ~y poorer cbMt lEe. MIC .. bcm ~y IIIIed

in FPLC in variousc;olrams.liUCb Q Mdto.Q HR SiS (RatIJay et al .• I~~ s.u..:-cbi et

11_ (999) ~ Q (Yeoman lad Edwll'dl.. 1997) ad Mono S Sol 10 (V ... et Il,

1994). Tbr tDOSl Q.1a1 •• iIClaly usat b)drophobic ad5crbena m: oc:tyt. (O·)..s phaiyl·

substibJted ~

43

T.blt 2.6 Combl ••• lon or purfficatfoD tKhaiques applied to purlflutlon or VlrloUI pruc",.cI

Microofpnl •. .:;;lD ____ _

Blcc,nt A!"flII8~'JfflltA~aJ,. A rtAroIuJ,1",- re'NJtt4IfM ~~.

811,;;/111$ "l'. ~719 Btldllll.f 1'. NCO(' 1 so

I14rlllll.f lip. SSRI B ".",ifts MK6-5

8, ~lJlJumuu MTIT 8-00104

B s"bII/ls rEil OIi~trc>pJra r",~oJttWWfl'" LlSM 1227 P,ffWl"IICu:h" ~. 1 ...... ~

PJrtldnmOlfa.l uru,i"IJJU MN 1 P. url",/"I)~u !'ST·n 1

C onttDlrI1lOU method

('\iH£):SO£ A,,1unc. (NHahSOa

(~H.t:50a (1I.1L):S04

l ",,.liIlnllnn

Uhraf1ltra1joa. {lIIIH.),sO. (N"'),,~), , JltratUhI1CJII

tNH..>:SO. {NH.),SC)i

COrtmm matrlces ______ ~_Clfc"'nce=_ __ _

OF.I\E ceJInlOlf. Stpbade), 0-100 Thanaam Ind Rljlumw. 2002 OEAE Sepbacy1. s,q,11.II:f)'1 200. Smaccbi It .1., 1 W'9 Phmyi SepbaJost. fPlC MODO Q HR. $/5 OEAE cd11dosc. a. ... ucin aprnIC OEAE Scpb&rosc: {,L·68. Of S~ C1..68. StphacrylS.200

lIutnddok·ToW1l1.,,, GC It.. 1999 Kurnlt ~l.l. am

DEAE Sqmade .. A~.sO. S,:phlt .. 61t SlnJh 1111.,20011 DE.O\I:: Scpham!tC CI..6R. Ku.mRt. 2002 CM Scptn.m..c Cl AiR. Septuaayl 5-200 CM Sepbotose fUr flow. DUll Sepharose fait flow. SephDuyl 5-100. 5-200. H)1lrupOObi~ iDteracrioD Pbeayllpl'Ollof:. Q-~opbanwc

SqWdex G-lOO ScpIwdp G-7S. 0I-ceU1lIoIe

nIlCYrTO)q)CtII65OC. 8 ... ,IIO)'OpCBt 650 M. PbcaylTo~16!oM. Tuyuparl MW·!OF ScpIuIda (;. HJO. DF.AF. cetlultlH SIIly)-Tnyopear! MOC, RlI'I)'J T 0)'UpCU1 650 M

HUlln. Clt .1,. 200J

Slnp et It, lOO I b

Adiaata),1tII cl al .• lOO) Kaa, 4,:' .t. 1999

0' .... et IJ.. 1997

B')'OUdb et .1.. 2000 0CiDO cc 11., 199'i1

P.fettdm,"IIftI.~ .... ULtraflhnuoa S- Sepbaroee flut now PO I () VI1"'1u&:J! t.lt al .• 200.t ~".a"a ""'~ (~HI.}SC)£ Q-Scpbarose. Sepbacryl 5·200 RomerD et 01,. 2001 ATa.' 2~19

"unal As~lI/uJ It",u, (UlRA 6.1) Ac.tone, tNH')lSO" DF.AF. SL-rhlLh:3l "25, SIlS·PAUh, Chlikrl6b.r11 4J1 itl .• 2000

elec:rroelution A,'(IH!rriltU., IlIm~dhI.J (NIt.):S04 Sephada 0-7~ nu~lc.llt al.. 2()()~ SC't'deJ'IJOrilllft apln '1"'"""'" (NH.),S( ) .. SqbacIts 0-7S. immobUjzed Lartlwrt* 11.. 199(1

p~u "'.000 pbeu)'l.~

4ctln.",ynlft ()m,Irf1\'tI.UI,,'h.mtt~, "r'f('U Ul1ratiUnt_ PbeuyS-ScpIIIroM Cl. "8. Slat C1 ,1.. 1994 TK·J (NHd,Sn. UEA!; SqJMcd SlrtpIO"'.~J 0"''''''' Aprolimn·.....-c PebnllC et al. 1999 SllTpru"'Yl"f/.t 11I~''ff/,.,rU Ultraftltration FPJ C F.aoao-pk Q. ScpL"IUIIC 12 Y I:UI1IIIft and l:dwllRla. t 991 Tht11fU1flctt'.mft.t'C ... liP. 1119 (NllehSO. DEAf ScpbnJte (1 .... 6R. r.cc et al .• 1996

Uut){-fO)'Opcart 6'0 M

Muuldl AIII'ff(J'~u~IJ'llm pIIJlIt/rlflJ UltrafiltraUou Scp~(i.7S O'""'*¥hy .n&1 MI:Kay. lW3 Cm/.#d4 r4""hwlyllr.'" Scpbacl)'l 5-200. DEAf 8J.I rota .1 111.. lOO 1

OfiAh·lJlclhyl .. tWDO c\byl, CM'ClrbolY It~lhyl

AfTmrt) pt«t,JUunO l~ • functJon of • wlult1c mlCfomolttwk UiprKt

pof,.'th.'"T.and a:u..-mh'-' Ih.a has t .. o f"u.:uoru.. (r) It conlairu an ilffiruty lipod

CJl"C'f~l) mote' rol~",akn' ~f~lc) and ~:I 11 (all be rreavrt*d m rr..ay

W8)". I e .• br champ' trl pit. tcmreratun: or iocuc Itrmllh Wl1h .hit ,C"Cmtqut'. the

JtpDd pol)1nCr l'- addcllO the nwynr soMlOh uadcr cundiliam fa\ICIfUIll bmd.1l1

of tM prulcln df In'ernr The hgant poJ)tn('f l.'> lixn ptt"lpdo1led, ..d tM

~I t~ tTmo\oCl! The prt'4em of m.~ Ij Iht-e tlu.i:'d from the pol)meT

uod.:1 urilable Condlh ... " and ~ po~ can M r«)dtd. An arkali~ pmlU5C

(MI\.tasc (rom 8 1i('lI#n"o"",,~), used .... washing powder Mdditjve. has brm

f'unf~ by.tlinl" Pfl'TapitlluCJn (he. et .1., 19-4iI11.

In Ikldiltoo 10 1be Doo\lC cbroma1ographlc techniquf.'5. A~l mlralirm is used

tor T4lrlld !lcparotlon ur rrutlcromolccules bu.!d on siu, Recently. many new agaTOSe

MW and mtJfC' flgid dnd (1'oss-hnked gell. !u(:b a5 St-pbacryl. SUPC:fU'Ie. ~upndn.

and 1 (\yoprarl art' .1'10 brlllg w.cd for punfiClIIDb ~ .. t") Th~' arc genmllly

u~ C'itbc.-r In the t'arh' ·fo·nuddll" "ugC' of purificlUoo (Chalaah.uti c1 at ~OOO) or

In thr fNlAt _~ O' pUlflCahnn (El-Sbanshoury et Al.. lq9~~ Lee cl al. 1996).

Mo1IJllf ~,l\anc.J!lC'1 "r:Ju~ method arc: 1hc '.",.a-Qlp.c.:ic) fur $u.MJi .. gptutnm. Md

.hll !he MSutd prole'" iCh lOO d,lWal

2.1 C ....... .maia ., ........

~ from iCWTal mICJDOIfpnt'lm hivr- heal ,..Iad,ed ~1kft5i\'d} aad

ba_ on IM_ prupcn.cs cM) are .-cl m \oIflOUt. ialuamo. fbc tmpOI'ba1

rropcma atC' ~ tn Tabk ~ 7, Jlo .. ne •• bri('f ICcount .. f IIIdlUdual

rn'P"12r§ " prncn1N m ~ foUo-.injt SCClJlJC.

4h

T.bl, 2.7 rroptrCfes of ..... , .......... 'rom dI"~real IIIkrobia .... rue -- - -

Mi~"... .. rll fcmp_ Swbll"I.lIf"'Ctn~y MW pi Olm prvp:rIiCII RelertllCe

~ 22hmt ("C) ~I 8te1"'. A '('tl/~/1',~ .Iu"''U/~. ",g .sS C""III1, U"A ... ,lIllln, 67 tu' ~n 1ftd~.lOOl

UO«) 11, L1zoolUoia Arf~roHrlfr 9,n~ 9~ 55 6O~17 tll.·1I#!.I ~~ilIIltI" ". 7U 71 ft..l, Sm~,;ld " 11.. t '99 "tcVff."1JII 94~M 1hId/~ It' J ij99 11.0 10 ('Man 19 ft. a. M .... IMl ~ JahnV<ltI)' and N.ik. lOO 1

~ kJl ... 'I' NOZ1 9.l .&1) '_Il n. ft.,.. IWUkar55111l1l Suandccpct .... 1909

.. 9Cfc &.rlllttt 'Cl. kSM·K.l'.&l I1 0 10 (:.em D.'. n". n.t.NIIIi~ $ita et a1.,l002 IMdlw lIP NCDC·IMJ 11.0. 1;2,0 'I);'S CIIIIaII. )"" ..... 1.: 2II~19 ft,., Sa&Mt lIP 10 pH II It .. "., I'll •• I"'"

p-llhrolnU*' "Uf',II.f ",. l'ti 179 '.0 75 Amcucm .t) .t,K ClL~ nbanca HlltldWk· T~"""III it .t .•

IhcrmolihlbUily 1999 (I~/'IfI,., .' !i!<iIU .0.0 .0 M.aetNln 29 t ... Ca'" enhances Sill", et al .• 200 1I

.hrnno~abili'y It. Iw~'", MTCC'IIOO16 IO.S )7 A,4Culnn ILl. nA Oetct&ent llaaerjce CIf 11 .• 1999

com.,.ablc • _",wm.'" HU 60 C'Win )0 01 8bd1-- .ad sns Ih, ..... ItlOl&;

-ttlble. "«tent Oap .. rut.. 1999 ~ •. ,...."u M"f) J 11.' !4-S5 p.Nt!rvtu"lh .... 2. D,I. C.l ' .iadepI!ndIm' Ku .... 2001

• " ..... ,lIJ H'l.O 55 l'.iII Jl 'I Dchairi III Kti"ily H.uaq n 11.. 200J If ... Ml/I. Itt.lt IU,O 00 C.-in I~ n,l, Stnm}1y JldiWllled Adin8t1lyllnl.' 11,. lOO.'

by C " MC'+. M:l· OII1l'JJr,,,,ltu IW Ml~ !O Cucin.IUUQI.H'UI. Z-' n,I. K'"Ia1 III t 199' t(,l~~'IIO'fJN1tI IIt1eOll. QIbon OS'" Ill? mono'lde .w.)!dn~w ,~ .. tl~. 9.0 ~o t"udn 1) 111 t: 0 r A RSDtaId 0,... G'I al .• I"' l·~.'

I~Sl'lld(lmllf1 /J,. 11, ~ ~, C'GlOU\ 311 n.s. Ot~1U111: IIOI~nl O¥~T1U III Ill" 1999 Q""',If/nosif PST-O I "lllhl~ S(f"'Mln fIlurt't.n t'II.f 9,~ 411 AfU4:1llI!:ln 66,5 n,., Rllmero ct 11" lOtH ATt.:c 2~419

had .( 1~'''''kJ .S J1 Na·astlllat~ • 37 n.s. Ch4knbof1j ot al .. 200() (inRA 6.2) ,ynthdK' tubitndn

(p -RltrOIniJidincs) Bt'QUll'f'Ul hclu lunll B"'.~ 25 A/UQJn.cl~ J13 7.S F.I ..... tnd UrtI: ud RIU'.lonO

anxk dclPdlllOn .... l1lrily

Aell ... .,(etn (HrJ.O\'UI ~,S )t,\) !O S)'nlheUa: m.~ 2D n.!.. VaI·/)'IIC ~lC)' SlCIkl f1 IL, , ~ .,u"t"I"~Ob·""1f n·t Srrc-plO"'Yf'n ')'II""IU I) ,/I 25 ('l.N.p.T~jrl-l,.,al,Siul~ 30.120 n. .. J'ctlnMc Of "I.. 1999

"ll!thyl~ St1'f'pl(lm,~'C''''v ("tldat 6.0 70 Azomcin 21 n.s. RftlllltlU11 1&1 S.tI)118 It al., 20(M

orawc IOI~lIh IUld ncMnaJ cllll.l.!rjOllIlI

TI'fff'mrlrJcI'nlJffl)'('f'I I HI ~~ ClQC:ln 31 C:alclum onhlU'lcc:4 L.co Cl .1.. J ~6 sl'. E19 Immr,u~b.biHI).

brold pH iflabllUy (5-12)

Ma.led, At#11'Oba:Jfd,,,. V.S -10.' ,u AzocolJ. u -ceseln 27 n.s.. ~)' IIId t.":Ka),. 1993 PIIllllla ... C .• ",dld" 4_~ 11.0 J7 Cucirl 30 .4.7 POIII t1 81 • lOO J Ctlst!!noJ.oju·u • NnI'PK1fil:'d

1.1.1 pH"" ........ ture kiadia

In prral. all curmuJy used dctft'JaUoCaDpldibIe proIease5 arc alkaline

md 1hmDo:sbIble in na1\lJ'C with a high pH opejmum (che pH of lauodry detergents

15 ~Iy in Ihr 1'3ItIt: 01 8.0 to 12.0) aDd ~ varyiag tboniOJlQh4lihes. at

lIundry ~ (~70=0· T'berebe ...... of Ihc UJmmC:rciaJly nailable

subtili5io.lype proteaSC$ an: a!Jo Kti....: in Ihc pH and imlperahlre I"IftGCS 8-12 and

S0-7~ rcsp«tiveJy (Table 2.1). In additiua. • ~l (rend in the dclagcat

mdu.ury is a ~l rw aJbline protease acti,,'t" at low washing tempenrurH;

for eaamplc.. Kaanase 4 marketed by Novozymcs - is active e\'m .. lemp:nhaR$ ~

low as JO-2OCC.

%.1.2 Erred of srabilizerlladdUlvf' ... d mtraJ ions

Some of tht majur commercial w.e!C of a1k.ali:ne prole05I:S nccCMitnlr high

temperatures and hence. improving the tbmnaJ &tability of the enzyme is dlllltinclly

Advantageous. ThermosLabilil), cln be enhanced eilhu by I:tddinS cel1ain IItllbilizer5

(PE.G. polyhydric aJcoboll. 51nrch ele.) '0 the reaction mixture or by nll,nipulating

the tcrti8J)' struc(UI'C or the et1z.yme by prott!rn engineering. A lhC1111OM.abiJiDtion

r-fJc.c4 of up to • 2-fokt laanK m che half·life of CuauiJitQ {ld/oUo protease I.

6S~ has bcaI reported by using pctlyhydric Il00bols. PEG and c.ueill .<ionD)cl et

aJ. 1992). l"be ion Cal- is also known LO p~y I map role in enzyme .a.bihlalton

by 1Dcrcasmg the ldi"ity and IhennaI 5tUiliJy of alkaline pI'OIC3W .. higher

t.aJIpcraNJ'eS (Kumar, 2002; Lee et al .• '996). 0Iher metal iOIlJ such 8$ 8a~ • Mn.'-.

Mar ..... Co:·, h r aad bl" are abo used for uInlizing pro4QSCS (John\"eSly and

Naik. 2001; Itanra)< cl .t. J99S). Tbc:sc IJJC'laJ ions protect the enzyJJIC agaiMa

IhmnI1 denaw:rlrion and pI .. y a ~itaJ role ID nwintaming cbe eclI\'C conflmllltioo of

49

-.tJU.J~ J'fOCtl.~ h.t1.~ broad subsnle speofied)' aDd ~ acn""l: ~Itit ~

...,..... 01 'I)'IIIhctlC sut.1ntcs aDd ...... pRtk1u H~vn. the btendurc

cmciusn.-er,.. "'gotm IhM tbry Hr mare ~T apmst ~ dsa .pmsa

a.ln:.~ IIctnoglobiD Of RS~ .Table 1 7). ~c.lhere arc-.pcrific ~ 01

alblilw P"*8a. ViZ-. (otlattenate. cla5lase~ ka'altfhD,C (Fncdnch C'l al .• 199'J, and

ans«1 CUfK]t~ pnMuc (Uru lad Ria. 2000). _inch an .ctI\~ aplQSl

~lfK pnMClft ~U~ Wl:b u rollagm. ebstm. knalift and CUI.ck Albline

prole.1Se5 ~ -110 specific IpmsI aroma.ic or bydrophobic ,,",ino Kid ~

su.:h.u t)~U)c" pftm,.altnme er If'UC'M at ~ rarboxyhc s.wk ,.(t.hc tJa\'il,r s1ct

(BMTrn. 200 1 )

2.1." KiDdic p.,.mrlrrl

Tu de\'C'lop In .:n/yme-b8.!lcd process. prior lnfomultinn about kmdle

PIlfBme1C1'S of Ihl: enzyme in quel1ion ill of U~I imporftincc. To br pre(l.e~

kmnlC' proptl1.~ like V_. K_ Kw and E. are imJ'Kll1ant. ~ln, not onJ)' ~1I1C­

~I'"=. but abKl r.ublb'lle IUld cn'Vlrt'JrtmaJl lpXifk. and "nowl~ of Ihoe is

alCDtW for designing tn7YmC reactor. ('If' quantifymg the arrhcathon. (If the

~me UDdc:T d'flnt'al t'."ndahU!I6. \'.an0\l~ comple:\t'l. ",z.. ClbIMn. azocasnn nc~

Md ,ynlhftlc subsualcs. "11 " p.nittoamhdr csun.~ utcd (or ddcnnltUnJZ,lunetK

)'Uahll1m for alkaline: pRJk'a5on, Thr 1o}"I'IIhrIX 'Ubt.lf"llCS eT muth ~ popular

the compla .U~1nIn fill dcfUUDf ~ and V_ as they 1ft ,on\'enttftl 'Kuu.-.

~1.. t....rdxt t1 .... 1996. fur an alblllK protea.'5e frf'l'h B- 1IW.141'C"JUU. lhr K.

for c.aK'in 4ro~ascd ",dh c~rtl@ mau.-.c iD V_. as the fC'acboII

lCniiJ*i&'Uff ..... u .. ncd from"~ ro6O"(" (Bcg et Al. ~OO~.) &1 CODCr.Dt. the K. and

V ~ ,Or _ .u..., IDr protr~ from 1tJft:(~ u,.,,-..IU' mcn::Med .,1It iIft ttKl'ra'C m

'cmpc·r.lfur~ ;; •• m n~c ~ iO'· ... t8ant'1)n" and BhMw:tw;)'a. lQOt,

Z.9 Apple.fio. of Protnsn

Proteases 0JIDJIi1ule a COIIIpIo. JroUP of en.zymn. ,..-Ilieh difTCT in rroperti~

AICb In subsuaIc spccirrly. ~ Ii1e md aIaI)1jc: nu_srn. pH Md ,~

of acti\'ity and 51ability profiJcs. Cccmuacial -PPIlcatioas of theJe eIIl)'IneI ID •

...,e of processes take Ihr advInIate of die unique physicaJ .. aIIalybc propcnies

or iadividuat procmt)1ic dU)UIt ~ They rcpresau OM of Ik duft J.ranl1fOUP of iJxbIJW au)1IIQ Md IIIIXl1WII fOr about 60% of tbe toW wor1ch,ide Ale or eDZ)1IIIS- Profc:IIn ~ • larJe varidy of applications. mBnty DJ 1bc dctcr,cat ....

food iIIdustrits. In view of the RCCOI Irmd or daoeloping «Olriendty ~

Ibc:y are an.isased 10 ba~ eJltCJ1S;VC applicatiom in kathcr bQtmc:n1 and in !IIeVer.l1

bi~atioo processes. The worldwide requircmrnt of cnzyJIJeS (or mdi\'idual

appJicaliom varies considerably, Proteases ;arc used eXlenSively in the

phannacc:uticaJ indu5try (or preparutiua uf medicines like oillbnerd5 for debridement

of wounds. in the food and detetaen' induMrie5.. prepared in bulk quaDlilics and u5.cd

as crude preparations. Whereas, those that are used in medicine are produced in !lmall

tllllOUnta bUI requi~ cxtcn.1iive pwifiCllion before they can be used. Some o( the

commercial pmleases an: listed iD the TablC' 2-1.

L9.1 Deie'lntlS

In spite of the fact tbal tbe ddetgcnt indumy is tht" largo.t sinale market

(or eazymcs at 25 • 10% of total us. dctai15 uf the CIlZ)'111Ie$ used and lite .... ys en

wbida dIcy are UKd. hav.: nudy been pubJished. Thm: aR lhn:c bawc ')'PQ of

~ wed 10 ddc:rgcutl: ~ amy~ and Iipases. Tht me of m1')'rbII:::fI

in detcrpt formulalicfts is now commo .. in dcw:loped counutn. WJth 0Yft h.U of

.U detc:rp:utS. pn:sClnly ~vadable contain mzyIIIC5- <ha lhc-~ 30 yean... !be

protfMrS in detapb ha~ dUlDpd rrom bring minor additi,,~ 10 tin .. IfIr k.t-y

insmliCDIS. Unlilladay. the largell ~ o(1he C'DZ}1DC IDIIkct has Ilftn held by

drtrrJent alkaline proteuQ. acU\'C and atable in t~ alkaline pH ~ (Gupla el

al .• 2002b).

51

ne ~ of Ihe tint enzymatlc detergent. "Bumus." dldeS back 10

19J 3. who CUlIiSkd of lOdium carboaate and a crude paocrcatic Q1rad. The finI

clttngad COIDintna .x bacIaW mzymr wu iDuoducuI in 1956 uodtt thr track

oame 810-40. la 1960. SO\'O IDdusIry AlS .ntrocIucod AblDott-. pmduced by

&r,/hu Jitrlw_i/onW; lIS commc:ttial name _'IS BIOTEX. Tb.is was foJl.owc:d by

w.uloUt" •• ck:Cc:fp'a1 tnIIdc by GisI·Brocadn.. L1~Jy. dtlCfFl proIl2JC5

ficcd • SdbKk in the- ntly 1970s. due: IQ uafavonbJc pubhcal)' v.iacn some

workers dcvck\prd an alJetg.ic n:actioo daring Ihc ..... lina or IheK nazymes. This

problem \\'~ .oly~ b)' tbc inuodudion ofdusl-f'n:e ~ulalcd produdA. Today.

detergcm enzymes accouaa for 89% of Ihe total protase sales la Lb: worl~ and a

sigRifM:lnt share of the rnarUt ~ captured by subtilisios aodIor alkaline pnMeasc::s

from many B«-iIIu:t ~prC1C'. The dclergenl enzyme market hili pown nearly 10-

fold during the past 20 years. In the I 980s and t..1ITly t 9905, tllc: .,lIJOf marlu:t ~~

(>SS%) of the dclcrgen1 clll)'mc was. held by Gist·Brocacb lO The Netherlands.

Genmcor lnternational in the Cmtcd States, Soh'ay in Belgium and Sbown·Kenko

inJapan. D~ginnin& in 1995. however. there "''"8$ consickrabJc need fur

Ji8lJorullizati{1O. in the dCI'-'1'gcrJl .:nzymc industry~ OWlng to the relativeJy hjgh cost

of manufacturing. coupled with increased ~ Iiom dClCf!ent manufacturers to

drive down raw tn.Ilerial tosls. GCDenCor Iatel'D.donal pwcbased the d.etergcnl

cazyme bU5iJ1C:$5O of GISt~s and SoI\1IY; and Novo :\ordin acquirc:d

Showl·Oenko·. ~I eIIZ)'IIIr buUneu. Today. No"'O NOldisk and Gcncnax­

lalc:mabooal are dx major arpphc1'5 of dctc:rgeut enzymes. supplying up 10 95% of

Ibe "...1 market or PfOlCa!U!S..

52

______ ---:::=-T_._bt~t .... 2 ..... 8-r:-.-"~~nlrd .. prodliftn of protnRl _Itb dldr tnd~ ume ·'·'.d~ ... m.. Orp.h.. t:"'''~c M •• u .... ".nr Lluadar, dC1tra.nt, Ah':IlIAllf

rDll1'7)"mc Uvdo-Uap BiopnlC WU:III

Faod IlIdoltr), Neutruc

Ptv~ Ondo a.p C:oro'- 70'9 E.rt&a.", .Ik ... rwattMot lFO

',.UlNS 'tl;~""tfo""'b World', fim mau JWOducc:d ~ rur dc~1 iftliua4ry NQ\'o Ngf~bk. t>eftlnork pH -N~uLraI.t mildly alkaJin. <pH 7.10). UlCd In f)C1Crltna. milk rmtctll modification 4 silk dqumm ....

AlkaletJ'hlllc 'adlhl,t .p. pH vptla:al I-Il. worU well "nttt, IMIt waahm. CMd.lticMl" lIMd fev \WIGI fimlhm,

AlkalaphUlc 14ttl'- IJ' HiIb pH AlempmfUre epti ..... (pH up It! 12) \Ked in ~~ cl wool ftnillWtc

rMlcin Clllincned \OWnI ofSariNJl« Prukia ~ ~ ..... s.n... .. 1lalN1.., III ..... ~iniq ~bI o1S~, UKd 1ft deeapats. deui",~

A.ctM Id law ~nQIa \Md .. OdtrptIU I)' low ... hl". , down "l 1 C).NC)

..

..

..

nexlpcr .. iua protcoe. .... ow. ~tr A. at hl,her """JlCrltUrtt .. S, IIf:.".t/u,.,..is If, .... hII/~. B","illu~ .p

lbtllrial , hc'wmjorwfu • rvhdlU , bc·4 ... p",.,

llitt'fJltnt Oocto Shll .. l. Jarln Dctnpt. cleani". N • ..., BludwmltlllJ. Jl$p!If1 tJckfJt1d Wu,,1 !i)'l'ldcr Bifl1l,,,ductll

(.~hlnl1

used in tm:wtr}. In &)~l mduftty ~tJn ftfbis4:uits. aKUn &. I:QOkin Complcs of cm &. c:n~tidat Used rar nten~ hydtul)'G oC prglcins lflCd" rood industryCmeat proceuU1l) Food Food Food

NoV(1 Ncml.lJk, Ocnmark

.. " Oodo Shu.'CI. Jlpan

Mob.,.. <.iumany ~~1. USA

t..llhrr ,ne .... na NOV<K!01 S No ... \) Unba~ Ecuyrnc (NUl!) NovftUme ~tI\,(l",lC I ()()

SUlluCQfAB \;Ova('CIf Wh Muacel. MIlIloIIII.11

rurllftel OX Pr!Jpu",~' Clan,near f'rOfOlI"e 899 MultU'act N4:LI~nl Muhlrltct r·)ooo Prow.6l 0rbcl .. n. O,'IIin, ...

Pmlntllcf

, ",b,,,,.. Foud

MICNbl.1 llsed ror soaklD& ll3lCd fur I.IIIhairing urh.icb A Qiu

1tm4e&'f Lipase mix for anaIllbM I'rypI'ft 11 ",'111;"'1), Serine prc4UU- Used for batint Wdn A ~i. m&kIlfkod &H .. ,.'I""

UIICd (\Ill' kid blu.1u.b. A w. Un for rt.ltarinl 'iIIU bhac he_ ~-"""'J" 1tCU .... pH

AlbJupbIU" ""'JII ... JP.

0Infl~ qlnoerad Donnr Ht", atlcahnc prokIK for dckrp&b

" I .. "/w u.pnitld 1ft

'Q~ fill.' "JI, Ollidatlvcly able: ddaJmt mQaIC lIIab .IkallM rroteue fnr law tcml'cracun: wuhln,

"A~tlrIDI Alkaline: prulalSC (or iDdustrial Prorcolyti. all:lt:rt.1 Neu1l'll protease for funcOonll J'IC'PCicI.IIc eta: So),. mtal Q1c) RM"lfll' MU.! .Ikalmc prutC&le for ptO'cin d!tatlon &.c:IIlri.J A'bUllt pmIaM: for JIIOIda d~ (..y Pd rwda, Alkllnrhilic kill., IP.

Ptcrtdl'l cftplCtCl'Cd "Id" of .Iblaph.il~ hdlJld •.

AJhJorhUI( I«IIhn 'P-

PmtaK P ... ."",.lIvz lIP. Diopruc COtltUluatt ,1, J.b,m" F.n,.ec:(I alkalIne f'tcICeUC ,- li""-'Ifonttu

C05O\Clic. pJllC'IDIteUtiQlI

hid....,.'

N .... IIIu.:hllmtnb. J,,,,"

" .. .. .. " l1 ... -broudes. the

N ..... t.Jt Oc~ ln~mltiolllll IDC .• USA

;' .. .. po .. .,

~".)' Enzyme. C1mbH. o.m.ny ..

I. N..- D;-~,"{eal.

J.".n 1!1~ IltYfb,'IfltI1*,f. USA

R .... nhpu." ... rl. pr!]" ... h cl.ullte CI')I"1 pm ..... t" ,.,'11, ptVI .. ~ rmIC_K

1lk.IIlY.t If

I'JtUtlill.um ••.• "r"'ltl"n.a Ancarch "'",dnlllrm4' ",rtlt'''",.fI RI.'lClU'Cb , , .. NUll to) Re.earc:h " , .. Mlt. Ihlo,,.II .. ' Re.teafth r'f"mr-Itl!l'" .. I""", Rann-b ------------------ --------------------------

••

.. .. .. Sp. Aldndl

Currml market trends and con~umcr needs arc influencing the

development of enzymes for dC1crgcnl appljcarioru;, ""ilh the emphasis OD enzymes

that h~ .... c improved performancelcos« nlUo!', increased activity and improved

compatibility " .. ·jth olhct detergent mgn.·dll'nls. In addition. enzyme supplien; and

detergent manufacturers are actively pur.;uing the development of new enzyme

acti"i1ies that address the cons.umer-exprcsscd need for improved cleaning. rabric

care and antimicrobilll benefits. However, apart from tJlelJ use 10 laundry

dctC'rgcnf~, they an: ullOO poputar in the tormulation of bnu~hold dilihlAi3~hing

detergents and both IIldu~trial and institutional cleaning dcC<,..,.gl.:."1lIS (Godrrcy and

West 19'J6~ Shl)well, 1999).

Con\tentionally, detergents have been used at elevated washing

temperatures, bu( at prcs~'1U there is considerable interest in the identification of

~)kallnc proteases, which are effective OVCf a wide tcmpcrutlln~ nmge. III addition.

the current consumer demands and lucll!3se.clllSt' of synthetic tibcrs. which cannot

lnlcratc high It.-mpcnltures. ha\'e changed wa.~hing hahits towards the llS~ of low

wushing lemperahu'es. This has pushed CtlLytnc manufacturers to look for novel

enzyme that can act under low tcmpcmlures. ~ovo Nordisk Bloindustry in Japan

has developed a detergent proteas.e called KannalOe, which kt."t.-p$ i1S high .;fficien<:y.

even at very low temperatures ( [0 :WOC).

There are many parameters involved in the selection of a good detergent

proteaseT such as compatibility with detergent components, e_g __ surfactanlS.

perfumes. oxidizing agcnl.s and blcach~~s (Gupta et al.. 1999: Kurnar et at. (998),

good ac1iviry at rcJcvunt washing pH and temperature, compatibility with the ionic

~trcng1b of tbe detergent solution, Slain degradation and removal pot<:nhtll. stability

and shC'lf life (Gupla cl H1. 2002b). In general, the m.ajority nf the ~olllrnc,.ciall)1

available enzymes are "Ol stable in the prc$eI1CC of bleaching/oxidizing ug~nls.

Hence. the laleF-t tfeud in cnLYmc-b&-ed de-tergents i~ the use ()f recombinant DNA

tcchrtulogy 11..1 produce biaengineered cnzymer. with beuer st~bilit)', Bh.:lu.:b and

56

OXKSalion 5labiJity has been intTOduccd tbroup me direcIcd ~ and

proem cnginecliog by the: replacement 01 cataiJlllDiao arid rcUdun (Bed. d al..

1993; Esadl et al.. J98S; wonr et at. 1996; Y., et aJ .. 20(0).

1..9.2 Food .... .....,.

TnditionaIly, microbia! pnltCMCS ba~ bccft nplot~ iD the: food

induslnn iD maay ways. JlKo ~ oC proteasn in lhr food InduS") d.dc:l hid t.o

-Miu;ty. T1Iq' ba~ Ilea IUUbDety mcd fOr various ~ MlCh u c~

makiog. baking. pic:pe,stioa or toy. bydrolysat~ and meal teDdai.zatioo.

2.'~ I Dairy iaduury

The major 8PJ'Jication of protcaJ4:S in the dairy industry i.. in th.:

manufacture of cheese. The: IrUJko(('lagulating enzyIIIeS fin into lhree main

c:alcBorir;s: (1) animal rennets., (it) microbial milk coagulanu. and (m) genelieat1y

e'lglneered chymosin. Both animal and m;crobial milk-coagulaling rmteaRCI\.

bclung to a class of acid upan8k proteases and have molecular weighb b~'IWCl!h

30,000 10 40.000 OH. Rennet C:l.tracted m:.m the fourth slomach of unweant'd

c:alYCs conlains the hlghe~ ndio of cbymos1n er£ 3.4.23.") to JlCPsm lC'h\'ity. A

world sbor1lgC of c:alr rcand due to die inC'RI,cd d .... -mand fCla- cheese product1on

has m..emined che search for .JtcmaU\lC microbial milk coapJ.u. £~'aWW:

roeatdI in thas area has ~lIcd in the production of C1IZYrP" ..... .e campldely

inxti\"3Ial It normal pastcu.rit.alion .~mperatuJa, and conWo \G)' - 1evd5 of

lJIDIIS)XCific prorcues. la c~se mlkin8. the plimmy rum:tioa of pmcra.~ is to

hydrolyle the speclfic peph* bond (Ihc PbcIOS-Mdl06 boad, to ~(' p-k­

casein IIDd macro pqMKks. ChymaI.in " prefmcd dIE 10 lIS high ipCCiflcify for

casein, v .. bidt is ~ble for it, c:a.«tJmI p:rfot .. JlUKe in c~ makinc,. The

ProlQSCS produced by GRAS (Genetically Rq2rdcd As Safc)-cleArt'd mkrnhef.

web as UftK:«" lIUdtd. IJadOIlS rublilts and E"dt)t!tia JHUDsuiC'Q arc gradually

57

rqUc,na tbytnalom ID cheeK maklJII.. 10 1988. ch)'lDl1l'm produced tbrouP rccarnbllWlI D7liA ICCbnolosY.·&J ft1'1t imroduced 10 ~ rut naJuaboIl.

WJxat flout tt • IDllJOI compont'tll of bahnc pnxnscs. h I.:oocaim ID

iosooJubk JlfUtc. c.lkd ,' ..... -lIkb di:1emuncs lhe Pf(lpCftla. 0' Iht' bKay

douglb. E.odo od e1optu1emun fNm hpttrglllw or;-=« ty,-c bccu ~ It)

modify .bea1 , .... by limibI PfC*Ol~. EtIzymMJc Ut".-man of ~ cbap facJ:.t.,t" .l~ tt..n.illlll.nct nYClwun, aod pcmutt the pra1lM;tiOh or. ",dft rarIF

of pnxlUCIS, I'M ldd1tion of prol~ mfuccs lhc miling time .rut I1$Ulb in

mara..'<1! loa.huk.&mr~ (R~ ,"ut. lWS)

2.9.2.3 Manll'.ture or MY prududa

So)'beAfI~ ~crvc Il~ I rich snurce offood. due tu their bjl!tb I:cnlLmt of good·

quabt}' rrotcin Pro1eAK'~ ha,,-e been used from lnCiml tim~s 10 rtrcpltC' ~ ~~

and ~lhC'r 10Y pnxlucts. The alblinc and neutra' protC.S~1 or ruupJ ongtD play ID

impNtant ro'~ tU Ihr prOCCIIJtftl of IiOY foatJC('

%.9.l." ".,blun'lII .r proet •• lIydrolyutes

"rbhnc protea.\C' Ia1.c bftn ~ m tht' prcp.amlOD U, ptOfaII

h:rdrolYNtH ot ~uah nulntioaal wluc. The prot~n l)drolysatcs cllmmoaly

~ from C~ID. .. be} proICUl uti lO)"J'I"OIC'ltI flDt m.a~ .pp&tQCxIG ID

hypolUapK: m(anl rood fonnullliom (AlQCIlUn Academy of pedi8Inc

'~nc 1989,. tor116cM,... of tun JUKeS or ""I dnnL.l. ~ IDIDUc.:tunq prOlrm­

ncb IhcnpeulK dteb ... hcakb producb ... f1.tvonnc 1IF"15. 1"M blner '-*

of protcUJ h)\Uul)1oaln l~ • UIIJ~ It.ima 10 tbcu WIC El food .cd talth Cola

prodliCl\ The mlcns.lt) or Ihc h1~ r1 propuftlCJUJ 10 the number of

bydrophohit.: Ul\.IDI.I Ktdt. ID tbc bydroJy5a1C rbc prcKDcc ". a proliDc residue ID

IM atlCtf of IM ptpcatk ..JIU COMnbu1es lit I~ btl1a'ncn.. The pqmd.a~ dW CM

5&

dca\le hydrophobic amino Kids IDd proline are ~-alaable in cXbi'1a1aa proldo

bydrolysaks.

~ (rom IIdic: IIrid badaia .-c aYaiIabk under the ndc

DIme Ddtitr3se.. C~ " ... hiP specificit)' for hydrophobic amino

Kick and hcuce~ has a pal poki'ItiaJ for dcbiflcriD&. A c.eful combination of ..

c:adoprotc:a5e for me priu_ y ..,.,Iym and MS amillOpqllidase for abc 5CCODIbry

~ is rcquiftd for the procIudion of. fuadioaal bydrol)Ale with mfw:cd

bidemeu.

8actcriaJ ncurral JIfOk8-~ are active in. narrow pH ran,;e (pH S,O 10 8.0)

• have relatiwly low tb.:nnololcrancc. Oue to their inlennedialc race or reaction.

nculral prOU:IISC5 gcneraie Ics5 binrm&!S5 iD bydruty-LCd food procenlt. than do the

animal proteinases and hence arc valuable (or use in tbe food indus.ry. NCLI.rlbe, •

neutral protease. la in!.cnsitive to the narural plant proteiDlse inhibitors and is

therefore useful in the brewing indlJslry (Rao et al.. 199K).

2.9.2.5 Meat teaderlZlltlon

Protc:aJiCS play. promin~'fI1 role tU Illeallendcrizatiou, C$pedully of beef. An

alkaline clasQsc (Tabgi eaaL (992) IOd thmuoplulic alkaline pratcue (WHIM C1

aL. 1992) ha\-"&: pnn'Cd 10 ~ 5t1tt'nsful and promising meat tmdcrizing auymn.

_.hey possess the ability 10 hydroJyzc COIIDCClivc: tissue protcins as well as mtlSC'Je

fibre JII'Ukios- The ICDdcrizatian process c.aa he achieved b}' spn .. ling cM

pcrA*mi CDZ)'IDC prepuatJon or by IDDIK'niioa ia aD CDl)'mt ~UIJOa aDdIor b)

Uljeamg ~ aJIICentntcd proIC.tic pre ..... ion ildO the bkNld s1JQm CIf me.t.

Soluble meat bydrolysalcs an also be dmved from Ian meal ~ ....

from ~ residues. after medwiaai cIeboaiJJs. by aolubmzation WIIb proteolytic

enzymrs.. Alcalue- has bca1 fOUDd to be I:bt mosl apprOJ"* n:zymc rn ltTmS or

COI\1. solubili1atian and ocber rdnaM 1.c1OB.. In an opmnizat process with

AlcaJascJ! at. pH or8.~ and tcmpc:rarure of5S-6{PC. a solubilization or 94~ was

S9

ICbint:d (O'Mean ed MUDJO. 1984a:b). The resuJuPlt nxat slUR)' is fUnher

pasrcuri7Cd to iOKIMlC the en~ and finds wide apphQtiaa in CII1DOd mal

products. ~ aDd sasooings. The clcmcd boacs 1nl)' aka IK- ~ as aD

excellent raw material f« the procIudion of gelatin.

Afblme proIea5eS can h)droJyze fllOIC'JIa from pa.as. r ... or _imlls 10

produce hydrolyu~. or wen-ddiaed pqMidc profile.. In additioa. proeeu. b)'droty<arr' hllVing angiokmin I-amvatiDg enzyme inhibitory idMty u.-ere

produad from sardine muscle by Ireatmalt ,,;dt • B. liclwrriflJl"lflU .Ibl,.

protease. These protein b~l)'5ates could be used df«tiveJy as • pbyNoIogic:aIly

fuoctional food Ib .. - plays In imponanl role iD blood pRSS&R reaulllion (M4Ibuj cl

ill., 1993).

KeralinoJ)'1ic Ktivity of alblint: protc:asc has also heen Hploited in the

production or prolCinaceous fodder from waste feathers or keratin-containing

materials. Alkaline prole.se! (812) from B. subtills and B. lich~nl/omJl.f PW&J

wa5 used for me hydrol}lfoIJ. of feacher kera~1O obtain D pro'cin COnCL"Db'atc ror

fodder production (Chcna et al.. 199!'; Dakv. 1990(1994).

2.9.3l..atller ladDft,,'

Alkaline I'fOk*oa wllh eluloJ)1ic and taalinOlytic activity can be used in

Icatber-pruc:cuiDg iDduMrin. Protean IIR used ror sdcclit.·e hydrolysU; of

noocolJaarnom oomlitueftls or the skm and for mDO,-.J of noofibrilJar proteios

sucb as &lbumms ...s Jlobulm. 1k (ODWCOliooaJ mdhods of ItAlhet procrssiDg

fnvofw hv.ardou5. chenualt ~ as .tium mlf •• which aaae probkm5 of

pulluIion aDJ dlluCDI dbplUl. Tb .... (or c:awiroomeoaal rU.!iOns.. the btoIreamcDt of

IcaIbcr USIQ& la atl)'mIhc IppfOICh is preferable aJ it offcn ,eventl ~

like Clby control. ~y "~k nxlUlCtion andecofrimdly (AndcrIm. 1998).

ProIeasc:s find thelr uw in EM waking. dehairinalDd batiDa stages 01

prcpcuing ski~ IIId hida. At pm;cnt. alkaline prolCDCi, wilh hydrated lime and

60

sodium chloride are uged ror deh.airi .... because the .JklJine condition,; enable the

swdJing of hair roots; and the ",b6oquent attack or pr'OCeaK on the hajr follicle

protdn aUClws easy ranoval of the hair. mu), .. in • sipliftt&nt reduction in ~

amount of 1QStcWMCf generated. Currentty. b')'pJiu U U5Cd in cambiDllion with

other #ltJd1hls aM AsJNrrgilhu pmteasa for bating. The selre."". of the enzyme

depends OIl its spedficity (or 1DIIbix proceinl IUch as eftilin _ kc:n&tin. and the

mIOUDl of enzyme needed drpmds on the type (If Je:atMr (soft or hud). No\"O

Nordisk mmufac:lares dilfeaeut prolcases and pnMCUe nux like Aquederm.

~ovocor S. NUE, Sovol.ime. Pyruc ~c. for use in difToenl foUges 01 ~

process ....

1.9.4 Te1lle iDdu*Y

Protease CllZ}1DQ In: used for wool pRKCainJ Md MgumminJ of 5ilk b

produciD!, SIDd washed effects on silk pnaenl~ Sericin" which as aboul25% oftbr

IOIaI -aght of raw ~Ik. cm--m lbc penpbay 0( the raw litk (&bus. thus providing

die I'OIIgh blcn of lhe Silt fibcB. Th,' !Cric:in is COII\'CDIJOnaIJy rem.nftl from die

IIII1Cr core of fibroin by ~ shrink--proor .. and twiAI-settq for the s.i1Jr

yams. usiag starch (KanebUa. 2000). The ~ is gennaUy C'xpm!ivc _

~rore an ~ method IUgnICd is the .. of C1lZ)'IJX pr~ such

as pure.e. for degu:mming the idk priM 10 dyeing. Treaunenc of Sia.ccUuJosjc

blcud is claimed to produce some unjque effects.. ProtealC'J an:.1so being \ReI w v.-ash down priori .. SCtNnS after &doe in Mda' to ieu~ the proteinKCOUl gums.

whicb are used for lhickming of priDriDJ ~ Bio-lIoninl aDd the cmdy

~laled pruoess of bio-polishing IUe perhaps allrKting most c~n' Idmtion in the

area of enzyme JII'O"ssing (Ramacbandnm and Kanhik. 20(4)

The wide diversity 3Dd specific-it)' of proleU1:5 11ft' used to grcaIldVlntage

in developing effectjVC' therapeutic IJ:.tCn'!.. Oral administration of prot~ frum

61

.~ otJ:M (luizym aad Nunu.:) his. been tiled ., • diFSli\~ I.d 10

C\XTett (C'fUJn l)1tC m.r,-mc dcfit1C1lC) S}"DdrUIDQ CkMtn4W ,ol~ er

JoUirutisw I~ uK\lm ,umbta.lUOU WUh brOld-5ptctrum ... btou~ mlbe lft'.uucm c.Jf burm ... \1WKDIk. An atpllJ"gilwc hdMnI fJum £. ,oIi b .cd 10 dai:aw

~ trom Lhc bluodstram 111 the \~ loons of ')mphOC)1lC I~kn­

Coll ..... ·tn .lIh .lk.11OC J"'C*&K al.Vlt)' ~ u..n:nmsJ'J UKd tar ~ ;ap,hcatioll'i~ In the prtparah'" of ~lO'Ao-rdease do:wIge (I'D. A new 1enU·.1UliDr

pr,,~ _uh baJh (ulUlCI"'~ oICU'lt! ~a~ produced b)' ."Jptrglllld 1lIP"

l.CF9. Th~ ~ftl)mr hydrulynd V2nou~ c;oII..,. 'YPft without anrino .ut R'k:iac

.1111.! liberated low m&.,IC"Cular .... ·~Ighr pcpljdt~ of potcnual tb~Qpa.IJC use

.R.vtbomruf ~ .1. 199'; t Som1&rI~', Ehut,w'-llSt • • ~tIOr. "I,ll hIp

CI"'IOlyJit a~li~ily Iiom 8ac,lIw .'rublill'f 316VJ. w .. ~ imrTMlhili7.l"d C'n a Nnll"Hc for

lb",rapcultc aJ'PJi'ltion In the tmltmcnc of bum5- and purulent .... mmcb. c.ubuncles.

funmclCi and deep ab:r.cr·ucs (Kudrya and Simoneruo. 1994). furthermo~

811(11/11$ 'Pp. ha\tc been ~cogniud •• beina lire 10 humans (deBnef and

DitkriC'Jucn. 19111 I and AI1 alb1i.ne pn'llcase ha\'mg fibrmolytlc leta\-1!)' h~ bcca

w.t"d u a1hrurnbolytk aecnt CKlm Cl -1.. J9%).

Smce the fin,a rtpon on ~~)zrd ~ ~)'hlhesti u''''1 dx

~Rl)'mltK rc:actton or bydrulyS$ (Beramann and fw.It;.:I-Coann. 1931).

* 1'fOCC&tCS ha\"e fnqutntly treu wed for pcpb4c S)~ «(1epo rf aJ . I997~

ISIlDD and NWJI"" 2000; Kue et a1 . Im Monhara. 198') Eftl)1lW~ peptide

,,~oft'cn ~ .ch .. ,....,. Ma cbcuuc:.al nclboft. e.,. rualDIl' caa be

pcrftmnaJ ~'CfC'O'IJ'C'C1tklll~' ad n:.-wsts do not R'qUJ~ $;« ·d,am Pf'*"roaa. mc~ lOlub-lhry 01 ~'ro1. su.b5truc~ «ifnftID' thcrmod~C' equtbbna 10

fll"~ ~j;" O\'Cf h)'drutYJtls. 1'bcre ." 1n5 Qftd for QpeMi\'f: rroccctin'·8~.

"");MlJC JohCUla • ., ~NS dElwc.al'lo. n:s.alIm, ID productlOh ,mu cornprtJtne

wnh ~ of tbt:lhlcal mcthdch (Monh .... 1987 •. Hownu. the- majOr 'umtatJoa

am..., ullj'fWltvn

fer die use of prolClSC in synthetic cbemiiUy iJ the Sl'R1II8Iy reduced lc:cj\1ty of the

aayme UIJda- anhydrous canditiam. Prot.cascs ha,,'e been used successfutJ)' for rhr

synIbesis of dipqJticb (Ram. d at. 1999) md lripeptjdt (So c1 al., MOO),

rqioselcc1M sugar et&aifladiaG (lti".. et aL, J 988) IDd cfia·~

bydrol~is ofpepbde csaas (Chco CC al .• 1991b). Anumbc:r ofreporu art' a\'&i"b~

CIa the use of a1bJiIIe pmcease Il1o peptide 5)'I1Ihrsi5 Dd thr 11¥lIutMln nf racanall::s

of amino acids.. The _urc IIId cypc of orpoX: soh-at have • 58'0lIl dTc:d OD

proteI5e .... ,ity in orpnic: solventl tK..w .. ~im Cl aL 1997), Subtilisin s.howrd

~foId higher acth.,ty in g]ycerol oompiWcd wilh edlylme glycol. N·

lIIdhyJfonnamidc, 1.2- and IJ·propancdioJ «('utro. 1999). The effm of~,

solVt.'1JI, medium and 5Ubstrllc mldtom., using a~hymotryp5in. suhlihsin BPftrf

and subtilisin Carlsbcrg from B .. utb"l,s SUiltn 72 on peplide synthcsi~ iD OI'paic

solvents was studied hy ;o\apshtmD el 1tJ. (1992) IlDd Gololobov et Ill. (1994), An

Industrial protease. Nc:ulnl,c (co-deposited with sorbitul UP eo ro.yamlde) for the:

Iynlbesis of several N°·"mtecled dipeptide derivatives in acetonitrile was used

(ClapeJ et aL. ) 997), The kinetic rt!lIoJucioll of N-protectcd amino acid C'!lcen in

organic solventS calalyzed by un indu!otriaJ alkaline protease, AIC'lII~efJ.1f· was

reported (Cben ct al.. I99Ja). Alblinr proe ... .hC ,,'as u.....ed thr the feflOlutlon ofOL­

phen)lalanine and ULllbmylglycane (SutatU et 11 ... 1992). Prottinue from an

eltb"t'mopbiJe. TIIt'nfflIS R141 A. rmmobdlud OD concrol1ed p.'lfC glass bc:acb. ...

usa! for prpbck ~ uAag cbe JoynthC5UO of Ih-Ala-Tyr·Nfh &.'io • model

SYSlCm.. Tbe ~ of. surfadUl-pmttasc c~cs _ • rtn\"rJ biocatalyst (or peptide

S)'Idhcsis in h)'dropbihc «pnic JOt\'Cl1ts ".-as described by ()bDkj et .t (2000)

Z.'.7 Silftt-ltKew!ry

Alblioe prOleUe!i play • crucial role m the biO~1ioWIlG or ~ X ·ray or

photographic films for sjJ~"CI' recovery. These v.'Mte films contain I s-!-O'~ ,ihu­

by wapt in their ~1aIin layer •• 'hi4:h &:10 be used as a good sou," or illvn- for a

varl~ of purposes. ConventiOMlty. (hi" lilver i~ tl."OO\'ettd by burnl,..., [ht" fLlms.

63

wluch causes UDdciirabk c:m.iromncolal polhJtion. Furthermore. bate: film made of

poIyaler QUDOl be I1:IClOYeftd asing this mdbod SiDce dx sitva is bound to

tdatin. .. is po55ibk to alrK1 sih:er from the proteiD layer by proCeoly1ic

be;UIiCiob. Eazya.tic bydroIysi5 of ~ DOt oaJy bdps in nncting silft!'. tu also Ibr rcqc:h.q o(poI)aIer film. AIblioe puc:as.c from B.. DbliJis dc:cotnpolClt

1be gdMia ia)'a' wiUrin IS -30 mia at SO-WC.ad relc:ued the silver fFuji-.a et

al. 1989; Nakibo&Iu et ...... 200J ).. AJbtine pnuase of /loc,/ha sp. 821-2 was used

for cbe cmymatic bydroI~ of tdaIia I&)'aS of X-ray fiIma 10 ~Jcue iiJ\'CI'

parIides (bIDb-a Cl .... 1993). The aibline pratCaSCS of &ldJlu ~p. 811

(Fajiwua et at... 1991) aad 8. cOQgultMu PIJ.. 77 wnr also cfficicna 'n dKnn.pMinl

die gelatinous coating on ~ X-ray fibm lium whlclt ~ ¥ItV" I.-ould be

fCCO\'cred (Gajju a al.. 1996).

2.9.1 Wade TreahMnt

ProtCUC5 wlubilizc proteinac:.eoWl wasle and thll5 help to IOWCf the

biological oxygen demand of aquatic !.)'SICD1S. Recently. the U)C of aJkahne

protease in the rDWlAgenlC'Iu of waste!. from various faod-proceu.lng induJl,ics and

11Ot1$ehold ICfivilies hAIl opL"ncd ur • DeW C'l1I in tbc- UJC' of protc::ues in \\'Ule

managmx:nt. The ule of kenilinnly1ic prolClSC for food and feed iDdUSlty 'ft'8ste.

(or degrndlns wa510 keratinous nwerial fiom pouJtJ) ~fUSe (lcbi& d a •• 200 I)

and as. dL-piIaIOf} IIgml lu mntt''e hair &om tht' drai_ (Tabma et al. 19CJ2, ~

ben rt'pOf'Ied. A fonnubtion coanmiDg proeeotytic C1IZ)'IIIn. &om Jl SllhtiJis. B .

.,ylollqu,.fad'-III and SIrqH~ ". and • disulr.cle rcducmg .I£CIIl

(diiogty.colllk). tbat mb.ncai hair dc-padatioa and hrl.". in dnrin& pipH clogrd

with hair-conlain'nl dcposiU. is cunmdy a\'ailllblr ia Ihc IDIIrkrt.. It was plqarcd

IDd pmalfd by (ienex (J1a1Mon et al... 19R5).

2.9.9 0tIIa Apple.do ..

Besides their indu!5lrial m;I medkinal applications. proIeUeS play m

dapottaDl role in buic research. Their selective peptide bond tJc::a,'ap is used in

64

the: elUl:id.tion of Aructu~~fun(."li(ln retaljoDihip. ;n the s)'nlhem of pcpbdeI ......

the lC'luaICing orproldnJ. In CSJCaC'C.1he wick spccificity oftbc bydrolytic «t,­ur prutr:un. rmck 8D calcu';vc applialion iD Cbe iOoIl ddergmt. 1caabcT. and

pbannacallicaJ iftdusuies and in Ihe JbUcIW'aI elucidalicm of proteins. ~

Ihrir r.ytllhdic eapKlliei are used (or the s)'lJdais of plOIcins..

65